Prof. Dr. ir. Peter Bossier · MGD1 mytilus galloprovincialis defensin 1 MGD2 mytilus...

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Transcript of Prof. Dr. ir. Peter Bossier · MGD1 mytilus galloprovincialis defensin 1 MGD2 mytilus...

Page 1: Prof. Dr. ir. Peter Bossier · MGD1 mytilus galloprovincialis defensin 1 MGD2 mytilus galloprovincialis defensin 2 MOPS 3–(N–morpholino) propanesulfonic acid n number of replicates
Page 2: Prof. Dr. ir. Peter Bossier · MGD1 mytilus galloprovincialis defensin 1 MGD2 mytilus galloprovincialis defensin 2 MOPS 3–(N–morpholino) propanesulfonic acid n number of replicates

Promoters: Prof. Dr. ir. Peter Bossier

Department of Animal Production,

Faculty of Bioscience Engineering, University of Ghent

[email protected]

Dr. ir. Nancy Nevejan

Department of Animal Production,

Faculty of Bioscience Engineering, University of Ghent

[email protected]

Members of the Examination and Reading Committee:

Prof. dr. ir. Jo Dewulf (Chairman)

Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Belgium; [email protected]

Prof. dr. ir. Mieke Uyttendaele (secretary)

Department of Food safety and food quality, Faculty of Bioscience Engineering, Ghent University, Belgium; [email protected]

Prof. dr. Daisy Vanrompay

Department of Animal production, Faculty of Bioscience Engineering, Ghent University, Belgium; [email protected]

Prof. dr. Annemie Decostere

Department of Pathology, bacteriology and poultry diseases, Faculty of Veterinary

Medicine, Ghent University, Belgium; [email protected]

Em. prof. dr. Patrick Sorgeloos

Department of Animal production, Faculty of Bioscience Engineering, Ghent University, Belgium; [email protected]

Prof. dr. scient. Thorolf Magnesen

Department of Biology, Faculty of Mathematics and Natural Sciences, University of Bergen, Norway; [email protected]

Dr. Jonathan King

Marine Centre Wales, Univeristy of Bangor, UK; [email protected]

Dean: Prof. Dr. ir. Marc Van Meirvenne

Rector: Prof. Dr. Anne De Paepe

Page 3: Prof. Dr. ir. Peter Bossier · MGD1 mytilus galloprovincialis defensin 1 MGD2 mytilus galloprovincialis defensin 2 MOPS 3–(N–morpholino) propanesulfonic acid n number of replicates

Nguyen Van Hung

The role of poly-β-hydroxybutyrate as protection against Vibrio infections in blue mussel larvae

Thesis submitted in fulfillment of the requirements for the degree of

Doctor (PhD) in Applied Biological Sciences

Page 4: Prof. Dr. ir. Peter Bossier · MGD1 mytilus galloprovincialis defensin 1 MGD2 mytilus galloprovincialis defensin 2 MOPS 3–(N–morpholino) propanesulfonic acid n number of replicates

Dutch translation of the title: De rol van poly - β - hydroxybutyraat als bescherming tegen

Vibrio infecties bij mossel larven

Cover page: Epifluorescence microscopy of microalgae and PHB- fed two day old blue mussel

larvae stained with Nile Blue A (Laboratory of Microbial ecology and technology, Ghent

University)

To cite this work:

Hung, N.V.(2016) The role of poly-β-hydroxybutyrate as protection against Vibrio infections

in blue mussel larvae. PhD thesis, Ghent University, Belgium.

ISBN: 978-90-5989-926-1

The author and the promoters give the authorisation to consult and to copy parts of this

work for personal use only. Every other use is subject to the copyright laws. Permission to

reproduce any material contained in this work should be obtained from the author.

This study was funded by a doctoral grant of the Vietnam government, Aquaculture

Biotechnology program, and VLIR-OI project (ZEIN2011PR389).

Page 5: Prof. Dr. ir. Peter Bossier · MGD1 mytilus galloprovincialis defensin 1 MGD2 mytilus galloprovincialis defensin 2 MOPS 3–(N–morpholino) propanesulfonic acid n number of replicates

LIST OF ABBREVIATIONS AND UNITS

i

A199 aeromonas media strain

AMPs antimicrobial peptides

ANOVA analysis of variance

AOD american oyster defensin

ARC4B5 PHB degrading isolated from blue mussel larvae

BLAST basic local alignment search tool

BRD brown ring disease

CA2 alteromonas sp.

cDNA complementary deoxyribonucleic acid

CFU colony forming units

Cg-Def1 Crassostrea gigas defensin 1

Cg-Def2 Crassostrea gigas defensin 2

CT threshold cycle

DGGE denaturing gradient gel electrophoresis

D-larvae two day old mussel larvae

DMSO dimethyl sulfoxide

DNA deoxyribo nucleic acid

DNA deoxyribo nucleic cid

ECP extracellular products

EDTA ethylenediaminetetraacetic acid

EEC european economic community

EPS exopolysaccharides

FAASW filter autoclaved artificial sea water

FAO food and agriculture organization

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LIST OF ABBREVIATIONS AND UNITS

ii

FASW filter autoclaved sea water

FNSW filtered natural seawater

FOS fructooligosaccharides

FTS flow through system

GI gastrointestinal

HPS heat shock protein

HSP70 heat shock protein gene 70

HUFA high unsaturated fatty acid

ICI Imperial Chemical Industries

LB35 luria-Bertani medium (salinity 35g L-1)

L-DOPA L-3, 4- Dihydroxyphenylalanine

MB marine broth

MCF medium chain length

MGD1 mytilus galloprovincialis defensin 1

MGD2 mytilus galloprovincialis defensin 2

MOPS 3–(N–morpholino) propanesulfonic acid

n number of replicates

ND not determined

N-FMLP N-formyl-methionyl-leucyl-phenylal-anine

NMS non-metric multidimensional scaling

NS not specific

OD optical density

OsHV-1 ostreid herpesvirus 1

PCR polymerase chain reaction

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LIST OF ABBREVIATIONS AND UNITS

iii

PHAs polyhydroxyalkanoates

PHB poly-β-hydroxybutyrate

PHB-A amorphous PHB

PHB-C crystalline PHB

PHB–HV poly-β hydroxybutyrate–hydroxyvalerate

PO phenoloxidase

ppA prophenoloxidase activating enzyme

ppm parts per million

proPO prophenoloxidase

PRRs pathogen recognition receptors

q-PCR quatitative polymerase chain reaction

RNA ribonucleic acid

RNS reaction nitrogen species

ROS reaction oxygen species

rRNA ribosomal RNA

SCF short chain length

SCFAs short-chain fatty acids

SEM standard error of the mean

SGR specific growth rate

SM Summer Mortality

SPSS statistical Package for the Social Sciences

TCD tissue culture dish

TOS transgalactooligosaccharides

UK United Kingdom

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LIST OF ABBREVIATIONS AND UNITS

iv

USA United States of America

UV ultra-violet

v/v volume per volume

VAR Vibrio anguillarrum related strain

VLB Vibrio-like bacteria

VIED Vietnam International Education and Development

β-HB β-hydroxy butyric acid

Δt rate of change

ΔΔCT relative gene expression

% percentage

µg microgram

µl microliter

µm micrometer

0C degree Celsius

cm centimeter

g gram

h hour

kDa kilo-Daltons

L liter

mg milligram

ml milliliter

mM millimolar

rpm rotations per minute

V voltage

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CONTENTS

v

CHAPTER 1: GENERAL INTRODUCTION ...................................................................................... 1

CHAPTER 2: LITERATURE REVIEW .............................................................................................. 7

2.1. The importance of aquaculture....................................................................................... 9

2.2. Bivalve mollusks in aquaculture .................................................................................... 10

2.3. Introduction to the Blue mussel Mytilus edulis ............................................................ 11

2.3.1. Taxonomy and distribution of Mytilus edulis ......................................................... 11

2.3.2. Life cycle of blue mussel ......................................................................................... 13

2.3.3. The importance of European mussel aquaculture ................................................. 16

2.3.5. Feeding behavior and digestive system of blue mussel ........................................ 18

2.4. The immune system of bivalves .................................................................................... 21

2.4.1. Cellular immune response ..................................................................................... 22

2.4.2. Humoral immune defense ..................................................................................... 25

2.4.3. Difference in the immune system between bivalve adult and larvae ................... 31

2.5. Constraints of bivalve aquaculture ............................................................................... 32

2.5.1. Bacterial diseases ................................................................................................... 32

2.5.2. Viral diseases .......................................................................................................... 35

2.6. Solutions to control diseases in bivalve larvae culture ................................................. 37

2.6.1. Water treatment .................................................................................................... 37

2.6.2. Antibiotics ............................................................................................................... 39

2.6.3. Immunostimulants ................................................................................................. 40

2.6.4. Prebiotics and probiotics ........................................................................................ 41

2.6.5. Antivirulence therapy ............................................................................................. 44

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2.7. The use of poly–β–Hydroxybutyrate (PHB) as a tool to control disease in aquaculture

seed production ................................................................................................................... 44

2.7.1. Introduction ............................................................................................................ 44

2.7.2. The production of polyhydroxyalkanoates ............................................................ 46

2.7.3. PHB production in Ralstonia eutropha................................................................... 48

2.7.4. Mechanism of antibacterial activity of poly-β-hydroxybutyrate ........................... 49

2.7.5. The immunostimulants properties of the Poly-β-hydroxybutyrate monomer ..... 51

2.7.6. The use of poly-β-hydroxybutyrate in aquaculture ............................................... 52

CHAPTER 3: APPLICATION OF POLY-Β-HYDROXYBUTYRATE (PHB) IN MUSSEL LARVICULTURE

.................................................................................................................................................. 55

Abstract ................................................................................................................................ 57

3. 1. Introduction .................................................................................................................. 59

3. 2. Materials and methods ................................................................................................ 61

3. 2.1. Crystalline PHB and amorphous PHB .................................................................... 61

3. 2. 2. Detection of ingested PHB by fluorescence microscopy ..................................... 61

3. 2. 3. Experimental design ............................................................................................. 62

3. 2.4. Isolation, culture and identification of PHB-degrading strains ............................. 64

3. 2.5. Molecular fingerprinting of larval associated bacterial community ..................... 66

3. 2.6. Statistics................................................................................................................. 66

3. 3. Results .......................................................................................................................... 67

3. 3.1. PHB ingestion by mussel larvae ............................................................................ 67

3. 3.2. Larval survival and growth .................................................................................... 68

3. 3.3. Isolation and identification of PHB-degrading bacteria ........................................ 72

3. 3.4. Microbial community analysis ............................................................................... 73

3. 4. Discussion ..................................................................................................................... 74

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CHAPTER 4: DOES RALSTONIA EUTROPHA, RICH IN POLY-Β HYDROXYBUTYRATE (PHB),

PROTECT BLUE MUSSEL LARVAE AGAINST PATHOGENIC VIBRIOS? ........................................ 79

Abstract ................................................................................................................................ 81

4.1. Introduction ................................................................................................................... 83

4. 2. Materials and Methods ................................................................................................ 84

4. 2.1. Bacterial strains and growth conditions................................................................ 84

4. 2.2. Amorphous PHB (PHB-A) ....................................................................................... 85

4. 2.3. Experimental mussel larvae .................................................................................. 85

4. 2.4. Blue mussel larvae challenge tests ........................................................................ 85

4. 2.5. Effect of β-HB on growth of V. coralliilyticus and V. splendidus ........................... 87

4. 2.6. Effect of β-HB on virulence factors of V. coralliilyticus and V. splendidus ............ 88

4. 2.7. Data analysis and statistics .................................................................................... 89

4. 3. Results .......................................................................................................................... 89

4. 3.1. Survival of mussel larvae in challenge tests .......................................................... 89

4. 3.2. Effect of β-HB on growth of V. coralliilyticus and V. splendidus ........................... 90

4. 3.3. Effect of β-HB on virulence factors ....................................................................... 93

4. 4. Discussion ..................................................................................................................... 99

CHAPTER 5: RALSTONIA EUTROPHA, CONTAINING HIGH POLY-β-HYDROXYBUTYRATE LEVELS

(PHB-A), REGULATES THE IMMUNE RESPONSE IN MUSSEL LARVAE CHALLENGED WITH

VIBRIO CORALLIILYTICUS ........................................................................................................ 105

Abstract .............................................................................................................................. 107

5.1. Introduction ................................................................................................................. 109

5. 2. Materials and methods .............................................................................................. 111

5.2.1. Bacterial strains and growth conditions .............................................................. 111

5.2.2. Spawning procedure and larvae handling ............................................................ 111

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5.2.3. Challenge tests ..................................................................................................... 111

5.2.4. Standardization of quantitative real-time qRT-PCR for mussel larvae ................ 113

5.2.5. Real-time qPCR standardization of primer concentrations ................................. 115

5.2.6. Total RNA extraction and Reverse Transcription ................................................. 115

5.2.7. Real-time PCR ....................................................................................................... 116

5.2.8. Real-time PCR analysis (2-ΔΔCT Method) ................................................................ 116

5.2.9.Phenoloxidase activity .......................................................................................... 117

5.2.10. Statistics and analysis ......................................................................................... 118

5.3. Results ......................................................................................................................... 118

5.3.1. Survival ................................................................................................................. 118

5.3.2. Design of immune gene primers for blue mussel larvae ..................................... 119

5.3.3. Impact PHB-A and/or pathogen on the expression of the antimicrobial peptides

(Experiment 2). ............................................................................................................... 121

5.3.4. Phenoloxidase activity (Experiment 3) ................................................................. 122

5.3.5. Time window of gene expression of AMPs (Experiment 4) ................................. 123

5.4. Discussion .................................................................................................................... 124

CHAPTER 6: GENERAL DISCUSSION ........................................................................................ 131

6.1. Introduction ................................................................................................................. 133

6.2. The effect of PHB on bivalve larvae and spat performance ....................................... 134

6.3. The effect of poly-β-hydroxybutyrate on the microbial composition in the digestive

tract of blue mussel larvae ................................................................................................. 137

6.4. Effect of poly-β-hydroxybutyrate on disease resistance of bivalve larvae ................. 138

6.5. Antimicrobial peptides (AMPs) immune response in bivalve mollusks ...................... 139

6.6. Induction of phenoloxidase enzyme activity in the early life stage of bivalve larvae by

PHB. .................................................................................................................................... 142

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6.7. The proposed model for future application of amorphous PHB in bivalve larvae

culture. ............................................................................................................................... 143

6.8. General discussions ..................................................................................................... 143

6.9. Further perspectives ................................................................................................... 144

Page 14: Prof. Dr. ir. Peter Bossier · MGD1 mytilus galloprovincialis defensin 1 MGD2 mytilus galloprovincialis defensin 2 MOPS 3–(N–morpholino) propanesulfonic acid n number of replicates

x

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LIST OF TABLES

xi

Table 2. 1: Life stages and characteristics of the blue mussel ................................................ 15

Table 2. 2: Classification of the hemocytes for different bivalve............................................. 23

Table 2. 3: Antimicrobial peptides of two closely related mussel species and their classes .. 28

Table 2. 4: Summary of defense mechanism recorded in blue mussel (Mytilus edulis) ......... 32

Table 2. 5: Experimental infection of bivalve larvae, juvenile and adults with pathogenic

Vibrio spp. ................................................................................................................................. 35

Table 2. 6: The evaluation of various PHB production systems reported for wild-type PHB

producing strains ...................................................................................................................... 49

Table 3. 1: Overview of the experiments ................................................................................. 61

Table 3. 2: Survival (%) and shell height (µm) of mussel larvae subjected to different PHB

treatments in experiment 1 and 3 (Day 14 post-hatching and Day 18 post-hatching) and in

experiment 2 (Day 10 post-hatching and Day 14 post-hatching) ............................................ 69

Table 3. 3: Settlement success (%) and shell height (µm) of mussel spat subjected to different

PHB treatments in experiment 1, 2 and 3 ................................................................................ 70

Table 3. 4: The final yield of mussel spat under different experimental conditions ............... 72

Table 4. 1: Overview of the different blue mussel larvae challenge tests ............................... 87

Table 4. 2: Swimming motility zone of V. splendidus and V.coralliilyticus after 24h incubation

on soft agar at pH7 and pH8 with different β-HB concentrations. .......................................... 93

Table 4. 3: The activity of different lytic enzymes (caseinase, gelatinase, lipase, hemolysin,

and phospholipase) of V. splendidus and V. coralliilyticus upon supplementation of varying

concentrations of β-hydroxybutyrate at various pH levels ..................................................... 95

Table 4. 4: Summary of the effects of β-HB on selected virulence factors of mussel larvae

Vibrio pathogens .................................................................................................................... 103

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LIST OF TABLES

xii

Table 5. 1: Summary of the challenge tests with V. coralliilyticus (105CFU mL-1) in the

presence of PHB-A (1 mg L-1) added 6 h before the pathogen .............................................. 113

Table 5. 2: Characteristics of the primers used for expression analysis with RT-qPCR ......... 120

Table 5. 3: RT-qPCR amplification efficiencies for all immune genes .................................... 120

Table 5. 4:The outcome of independent t-test between challenged and un-challenged mussel

larvae ...................................................................................................................................... 124

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LIST OF FIGURES

xiii

Figure 2. 1: World capture fisheries and aquaculture production .......................................... 10

Figure 2. 2: Global bivalve mollusk production by species ...................................................... 11

Figure 2. 3: Blue mussel Mytilus edulis .................................................................................... 12

Figure 2. 4: Worldwide distribution of bivalves. Mytilus edulis lives in the Arctic regions and

European waters ..................................................................................................................... 13

Figure 2. 5. Blue mussel larvae development .......................................................................... 14

Figure 2. 6: Schematic illustration of reproduction cycle ....................................................... 15

Figure 2. 7: Production of the blue mussel, Mytilus edulis in the world and in Europe ......... 16

Figure 2. 8: Production cycle of Mytilus edulis ........................................................................ 17

Figure 2. 9. Digestive system of M. edulis: Illustration of the stomach and digestive gland .. 19

Figure 2. 10: Schematic illustration of filter feeding process ................................................. 19

Figure 2. 11: Comparative sizes of suspension-feeding bivalves and their potential food

(seston). .................................................................................................................................... 20

Figure 2. 12: Mechanisms involved in the bactericidal activity of bivalve hemolymph .......... 22

Figure 2. 13: Schematic illustration of phagocytosis ............................................................. 25

Figure 2. 14: Diagram of the major reaction cascades from the initial pathogen contact

through oxidative killing. ........................................................................................................ 30

Figure 2. 15: Pseudomonas putida cells accumulating PHB inclusion under electron

microscopy ............................................................................................................................... 45

Figure 2. 16: General structural formula of poly–β–hydroxybutyrate .................................... 45

Figure 2. 17: A schematic representation of the biosynthesis of PHA in bacteria ................. 47

Figure 2. 18: Metabolic pathway for microbial production of (R)-3-hydroxybutyric acid ....... 48

Figure 3. 1: Light (A and B) and epifluorescence microscopy (C and D) images of starved

larvae (A and C) and larvae fed with Nile Blue A stained PHB for 2 hours (B and D) .............. 67

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LIST OF FIGURES

xiv

Figure 3. 2: Size class distribution of mussel larvae on day 14, subjected to different feeding

regimes in experiment 2 .......................................................................................................... 71

Figure 3. 3: NMS ordination based on Bray Curtis similarities for the bacterial communities

associated with the larvae subjected to different feeding regimes in experiment 1. ............. 73

Figure 3. 4: NMS ordination based on Bray Curtis similarities for the bacterial communities

associated with the larvae subjected to different PHB concentrations in experiment 2. ....... 74

Figure 4. 1: Blue mussel larvae after Lugol staining (x 400). Living larvae (A) are completely

dark while partly (B) or entirely (C) empty shells are counted as dead larvae. ....................... 87

Figure 4. 2: Survival of blue mussel larvae challenged with V. coralliilyticus or V. splendidus at

105 CFU mL-1 under different experimental conditions. .......................................................... 92

Figure 4. 3: Growth curves of V. splendidus and V. coralliilyticus (OD600) in LB35 at pH7 and

pH8 with different concentrations of 0, 1, 5, 25, and 125 mM β-HB. ..................................... 92

Figure 4. 4: Biofilm formation (a & b) and exopolysaccharide production (c & d) of V.

splendidus and V.coralliilyticus. ............................................................................................... 99

Figure 5. 1: Experiment 1. Survival (%) of mussel larvae after 48h exposure to V. coralliilyticus

(105 CFU mL-1) in the presence or not of PHB-A ................................................................... 119

Figure 5. 2: Experiment 2. Expression of immune genes (A) mytimycin, (B) mytilinB (C)

lysozyme, and (D) defensin .................................................................................................... 122

Figure 5. 3: Phenoloxidase activity (PO) in the mussel larvae collected after 48 h exposure to

the different treatments ........................................................................................................ 122

Figure 5. 4: Expression of immune genes (A) mytimicin, (B) mytilinB, (C) lysozyme and (D)

defensin in challenged mussel larvae with V. coralliilyticus at 105 CFU mL-1 ....................... 123

Figure 6. 1: Schematic overview of the study on the mode of action of PHB in mussel larvae

culture. ................................................................................................................................... 134

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1

General introduction

CHAPTER 1: GENERAL INTRODUCTION

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2

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Chapter 1 General introduction

3

Aquaculture production is expected to play a crucial role in meeting the growing demand for

aquatic products, especially since capture fisheries have markedly stagnated with little

likelihood of increased production in the foreseeable future. Currently, aquaculture is the

fastest growing sector in the food production industry with an average yearly growth rate of

more than eight percent over the past two decades (FAO, 2014a). In 2011, total global

aquaculture production of mollusks reached 14.4 million tonnes, accounting for 22.9% of

total aquaculture production of 62.7 million tonnes (FAO, 2013).

Bivalve mollusks are an important global food commodity (Sauve, 2013). Natural populations

cannot meet the increasing demand for seed due to overexploitation, which has led to the

development of aquaculture techniques (seed capture, hatchery practices) (Avendaño and

Riquelme, 1999). Hatcheries have several advantages, such as the possibility to satisfy seed

requirements out of the natural growing season, the supply of genetic strains with improved

biological characteristics or the supply of new bivalve (exotic) species. Nevertheless, the

culture of bivalves in hatcheries is frequently affected by severe disease outbreaks, mainly

caused by bacterial infections of members of the genus Vibrio, resulting in the loss of

complete batches and compromising the regular production and the economic viability of

the industry. There are many descriptive studies detailing these outbreaks, but only a few

focus on the control of microbiota. The unique characteristics of bivalve aquaculture must

be considered in the design of improved hatchery systems. The classical treatment is

directed towards the complete removal of bacteria from the culture seawater. However, this

objective is on the one hand unfeasible, because the cultures are not axenic, and on the

other hand undesirable since some bacteria enhance larval development (Prado et al.,

2010). Larvae and bacteria, including both beneficial and potentially pathogenic ones, share

a common environment. The filter feeding behavior of larvae increases the strong influence

of these bacterial populations on the hosts.

The particularities of bivalve aquaculture in hatcheries must be considered to design

methods of control. A number of methods have already been developed to control the

proliferation of pathogens and to maintain a healthy microbial environment in aquaculture

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Chapter 1 General introduction

4

systems. Classically, the water is subjected to treatments with the aim to reduce the

associated bacterial population. Decantation and subsequent filtration of natural seawater

are common practices, while additional procedures such as ultraviolet radiation or chemical

treatment have also been employed to attain the complete elimination of harmful

microbiota from the culture water. However, this objective is not practical as bacterial

populations, or, at least, part of them, have a beneficial effect on the larval development

(Hidu and Tubiash, 1963).

While vaccination has been a major success in fish culture (Romalde et al., 2002, Herrera and

Toranzo, 2005), vaccination procedures cannot be applied successfully to mollusks because

they have only an innate immune system and lack an adaptive immune system. However, a

number of alternative strategies have been tested to enhance larval cultures performance of

bivalves by modifying the microbiota or by interfering with the innate immune system.

Among these are the use of probiotics (Douillet and Langdon, 1994), immunostimulants

(Bachère, 2003), and antimicrobial peptides (Defer et al., 2009). However, the

implementation of these alternative techniques should be based on the understanding of

the mechanisms involved and their putative consequences (Marques et al., 2005). Therefore,

new solutions need to be developed and validated for the control of bacterial diseases in

larval cultures of bivalves in hatcheries.

Poly-β-hydroxybutyrate (PHB), the simplest and most common member of the

polyhydroxyalkanoates (PHAs), a class of linear polyesters produced in nature by bacteria

out of sugar or lipids, is considered to have a high potential in human medicine as a

degradable implant material because of its non- toxic nature. (Freier et al., 2002). Several

studies have demonstrated that PHB might constitute an ecologically and economically

sustainable alternative strategy to fight infections in aquaculture (Defoirdt et al., 2006b), It

has been suggested that, PHB can be degraded into β-hydroxy short-chain fatty acids

(SCFAs). SCFAs specifically downregulate virulence factor expression and positively influence

the gastrointestinal microflora of the host (Defoirdt et al., 2009). Currently, there is

considerable interest in SCFAs as bio-control agents in animal production. Therefore, PHB

could be a potential candidate for the biological control of bacterial infection in bivalve

larvae culture as it could be a source of SCFAs after depolymerization of particulate PHB,

provided the latter can be easily accumulated by the filter-feeding mollusk larvae.

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In this study, the blue mussel Mytilus edulis was selected as a model species because it is an

important aquaculture bivalve species in Europe with high production (Lobel et al., 1990)

and because of the relative easiness of handing larvae of this species under lab condition.

The overall objective of the present thesis is to investigate whether the application of PHB in

larval cultures of the blue mussel, M. edulis, can increase survival. It has been suggested that

PHB can boost survival in aquaculture species by acting as a bacteriostatic compound and/or

by stimulating the immune system. Yet up to now it has not been verified where PHB is also

instrumental in sustaining a high survival in mollusk larviculture (chapter 3), neither had it

been verified what the mode of action of this compound could be. Possible mode of action

could be a direct inhibition of pathogens (chapter 4), an impact on the diversity of the

microbial composition in the intestinal tract of the larvae reducing negative host microbial

interactions (chapter 3) but also a potential stimulation of the innate immunity of the

mussel larvae by (chapter 5).

As a general introduction to the results chapters the production of bivalve mollusks in

aquaculture is reviewed. The advantages and constraints of larval production are described,

jointly with an overview of the most common diseases in hatchery-reared bivalve larvae and

common strategies to control pathogenic bacterial infection (Chapter 2).

- In the results chapters, the beneficial effect of PHB is investigated together with a

verification of some putative mode of action.

- In order to do so the effect of PHB on blue mussel larvae was identified (Chapter 3). This

was achieved through the subsequent 3 experimental approaches:

By evaluating the blue mussel larvae’s ability to ingest PHB using fluorescent

microscopy of the gut. Different forms of PHB (crystalline PHB vs. amorphous

PHB Ralstonia eutropha containing 75% DW of PHB) were supplemented in

the culture water.

By determining the optimal PHB dosage for mussel larvae culture in a large-

scale experiment where three dosages were tested. Larvae survival, and

growth and settlement were the monitored parameters.

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By verification of the effect of PHB on the diversity of the microbial

community composition in the digestive tract of mussel larvae using DGGE

(denaturing gel gradation electrophoresis) on amplified 16S rDNA fragments.

- In addition the antimicrobial activity and the protection offered by PHB-containing

bacteria against different pathogenic Vibrio strains was verified using in vivo

challenge tests with mussel larvae in a nearly-gnotobiotic system. Furthermore, to

document a potential mode of interaction between PHB and a particular Vibrio

strain, the effect of hydroxybutyric acid on the growth and virulence factor of Vibrio

pathogenic bacteria at two pH levels (pH7 and 8) was studied by in vitro experiments

(Chapter 4).

- Finally, to monitor the immune reaction in mussel larvae in attempt to understand

why PHB enhances their survival of mussel. The innate immune gene expression and

phenoloxidase enzyme activity of mussel larvae challenged with Vibrio coralliilyticus

was analyzed. In addition the production of selected antimicrobial peptides during

specific bacterial infections was monitored (Chapter 5).

- Finally an overall discussion is presented, the main conclusions are drawn and

perspectives for further research are elaborated in Chapter 6.

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Literature review

CHAPTER 2: LITERATURE REVIEW

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2.1. The importance of aquaculture

Aquaculture comprises all forms of culture of aquatic animals and plants in fresh, brackish

and marine environments (Pillay and Kutty, 2005). The role of aquaculture in ensuring a

consistent supply of aquatic species for human consumption cannot be overstated. Seafood

production, especially fish, is important for future global food security. Fish, as well as other

aquaculture products such as mollusks and crustaceans are nutritionally very important to

humans, as it provides all essential amino acids and is an excellent source of proteins, trace

elements and polyunsaturated fatty acids (Kris-Etherton et al., 2002). Currently, we face an

increase in global population that is expected to grow by another 2 billion to reach 9.6 billion

people by 2050 with a concentration in coastal urban areas (Glenn, 2014). The world fish

consumption per capita apparently increased from an average of 9.9 kg in the 1960s to 17.0

kg in the 2000s and 18.9 kg in 2010, with preliminary estimates for 2012 pointing towards

further growth to 19.2 kg (Belton and Thilsted, 2014). Fish consumption is not equally

distributed geographically. For example, in 2014 fish consumption in Africa (Congo, Liberia)

was 9.7 kg/person/year while fish consumption in Vietnam amounted to 38.3 kg/person/

year. The driving force behind the impressive surge has been a combination of population

growth, rising incomes, and urbanization (resulting in better distribution channels)

interlinked to the high expansion of fish production.

According to the available statistics collected globally by FAO, world aquaculture production

attained another all-time high of 90.4 million tonnes (live weight equivalent) in 2012

(US$144.4 billion) (Figure 2.1), including 66.6 million tonnes of food fish (US$137.7 billion)

and 23.8 million tonnes of aquatic algae (mostly seaweeds, US$6.4 billion) (FAO, 2014b). Not

only is aquaculture important for future global food security but it is also the most

sustainable form of animal production. With growing global population and declining natural

resources aquaculture is increasingly being viewed as the most environmentally sustainable

form of animal production. The food conversion rate (FCR) of fish production (between 1

and 2 in aquaculture setting) is less than all other production animals, including cattle (FCR

>6), swine (FCR < 3.5), poultry (FCR < 2), while bivalves being primary consumers, are even

more efficient (FAO, 2012). For bivalve spat a FCR of 0.4 is generally accepted in hatcheries

(pers.comm).

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Figure 2. 1: World capture fisheries and aquaculture production (FAO, 2014b)

The global food sector is facing various challenges related to the recovering economy and

demographic issues, including growing urbanization. In particular, urbanization will bring

with it changes in lifestyles and consumption patterns. In combination with income growth,

it may accelerate the ongoing diversification of diets in developing countries (Garrett and

Ruel, 2000). Therefore, demand for fish products is expected to continue to rise in the

coming decades. However, future increases in per capita fish consumption will depend on

the availability of fishery products. With capture fisheries production stagnating, significant

increases in food fish production are forecasted to come from aquaculture.

2.2. Bivalve mollusks in aquaculture

Bivalve mollusks (oysters, mussels, clams, and scallops) form a significant part of the world’s

fisheries production. Bivalve mollusks represented almost 10 % of the total world fishery

production, but 26 % of volume and 14 % in value of the total world aquaculture production.

World bivalve production has increased substantially in the last 50 years, going from nearly 1

million tons in 1950 to about 13.9 million tons in 2010 (FAO, 2014b).

China is by far the leading producer of bivalve mollusks, with 10.35 million tons in 2010,

representing 70.8 % of the global molluscan shellfish production and 80 % of the global

bivalve mollusk aquaculture production. All Chinese bivalve production is cultured. The other

main bivalve producers in 2010 were Japan (819,131 tons), the USA (676,755 tons), the

Republic of Korea (418,608 tons), Thailand (285,625 tons), France (216,811 tons), Spain

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(206,003 tons) and Chile (300,000 tons). By species, the bivalve mollusk production from

aquaculture in 2013 consisted of 37 % (clams, cockles, arkshells), 36 % ( oysters), 13 %

(mussels) and 14 % (scallops, pectens) with an impressive growth in the production of

oysters, clams, cockles and ark shells since the early 1990s (Figure 2.2) (FAO, 2015)

Figure 2. 2: Global bivalve mollusk production by species (FAO, 2015)

2.3. Introduction to the Blue mussel Mytilus edulis

2.3.1. Taxonomy and distribution of Mytilus edulis

Mytilus edulis is a taxon belonging to a group of three closely related taxa of blue mussels

known as the M. edulis complex (de Meeiis and Renaud, 1997). This group comprises of M.

edulis, M. galloprovincialis and M. trossulus. The taxonomy of these species is based on their

morphological and genetic differences, and they can hybridize when present in the same

locality (Gosling, 1984, McDonald et al., 1991). Linnaeus, 1758, described the scientific

classification of Mytilus edulis (Figure 2.3) as:

Kingdom: Animalia

Phylum: Mollusca

Class: Bivalvia

Scallops, pectens

14%

Mussels13%

Clams, cockles, arkshells

37%

Oysters36%

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Order: Mytiloida

Family: Mytilidae

Genus: Mytilus

Species: Mytilus edulis Linnaeus,

English name: Blue mussel

Figure 2. 3: Blue mussel Mytilus edulis

Mytilus edulis has a wide distribution pattern, extending from the Artic towards the mild

subtropical regions. In European waters, blue mussels are distributed from the White Sea in

the North (Russia), over the North Sea (The Netherlands and Belgium) and the Atlantic

Ocean (the United Kingdom and Ireland) to the Atlantic coast in the South (Southern France)

(see the red circle in Figure 2.4) (Gosling, 2003, Beaumont et al., 2007, FAO, 2016). They

occur most abundantly in the tidal and sub-tidal zones, ranging from the poly-haline to

mesohaline estuarine environments. They tend to proliferate on the rocky shores of the

coastlines, bays, and river mouths which usually show high levels of nutrient loads (coming

from the land), a necessary condition for phytoplankton blooms (Newell and Moran, 1989).

Salinity has relatively little effect on growth and survival of both larvae and adult in contrast

to other exogenous factors such as temperature or food availability (Widdows, 1991).

Adult mussels have been reported to survive to harsh winter periods even when the sea is

covered by a thick ice layer (Hatcher et al., 1997). Low temperatures however, significantly

influence the growth and the development of larvae, particularly temperatures lower than

160C. The strongest growth of larvae is obtained at temperatures between 16 -220C but this

range varies among larvae originating from different geographic regions (Widdows, 1991).

Brenko and Calabrese (1969) also recorded optimum larval growth at 200C within a salinity

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range of 25 to 30 g L-1. Decreases in growth rate were experienced at both 100C and 250C,

especially at low (20 g L-1) and high (40 g L-1) salinities.

Figure 2. 4: Worldwide distribution of bivalves. Mytilus edulis lives in the Arctic regions and European waters

(red circle) (Gosling, 2003).

2.3.2. Life cycle of blue mussel

The common blue mussel (Mytilus edulis) has several stages in its life cycle (Figure 2.6). The

first stage occurs when two adult mussels produce and release a large amount of sperm and

eggs into the water where the fertilization takes place. The sperm is released before the

eggs, in fact sperm triggers the release the eggs (Gosling, 2003). Females can produce up to

8 million eggs per individual and for each egg there are about 10 000 spermatozoa released

by males (Zaidi et al., 2003). The spawning season depends on different factors such as the

environmental water temperature, food availability, salinity and occurs from early spring

(April) till the end of summer (September) (Gabbott, 1976). Hatching usually occurs on

average 24 hours after fertilization, forming a ciliated embryo (trochophore larva). The

ciliated velum serves both to propel the larva and filter food particles. These embryos

continue to undergo a series of different veliger stages as explained below. After 2 days of

hatching, trochophore larvae have developed the first larval shell called prodissoconch I

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secreted by the shell gland and the mantle epithelium. Based on the shape, larvae at this

stage are also known as “D-larvae” or “straight hinged” larvae. As the veligers continue to

develop, the second shell called prodissoconch II is secreted by the mantle. Larvae at this

stage are called a veliconcha and distinguished themselves from the D-larva by the

development of umbo on the top of larval shell (Figure 2.5).

Figure 2. 5. Blue mussel larvae development

This larval stage can last for a week depending on environmental conditions. During that

time it grows in size and develops a pair of photosensitive pigmented eye spots and an

elongated foot with a byssal gland, and is termed the pediveliger. Once entering the

pediveliver stage it starts searching sites and substrates for settlement. Once settled by

secreting the byssal thread to attach to the suitable substrate, the larvae lose or reabsorb

the velum and undergo metamorphosis stage to develop into spat which resemble the

adults in shape (Newell and Moran, 1989). The life stages and characteristics are presented

in Table 2.1.

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Table 2. 1 Life stages and characteristics of the blue mussel (Newell and Moran, 1989)

Stage Size Age and characteristic

Fertilized egg 68-70 (µm) 0 – 5 h non motile

Trochophore 70 -110 (µm) 5 – 24 h ciliated and motile

Veliger larva Up to 35 days; start feeding using velum

- Prodissoconch I 110 (µm) Straight – hinged shell

- Prodissoconch II 260 (µm) Occurring umbo

- Eyed larvae 220 – 260 (µm) Development of eye spots

- Pediveliger 260 (µm) Development of food

Spat 0.26 – 1.5 mm Up to 6 months, temporarily attached to

filamentous substrates

Juvenile Up to 2 years Up to 2 years, sexually immature

Figure 2. 6: Schematic illustration of reproduction cycle (Waikato, 2013)

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2.3.3. The importance of European mussel aquaculture

There were reports in France in the 13th century of cultivation on wooden stakes (Garen et

al., 2004). Blue mussels are also among the top 5 economically important species that

contribute to the worldwide production of bivalves. In 2013 global production of blue mussel

M. edulis was 197.831 metric tonnes (mt) of which 184.873 mt (93.45%) was produced in

Europe (FAO, 2014b).

Over the last 50 years, the major producers of blue mussel M. edulis have been Denmark,

France, Ireland, the Netherlands, United Kingdom, Sweden and Norway (Lee et al., 2012)

(Figure 2.7) while M. galloprovincialis (Mediterranean mussel) has mainly been produced in

Spain and Italy (Smaal, 2002)

Figure 2. 7: Production of the blue mussel, Mytilus edulis in the world and in Europe (Lee et

al., 2012, FAO, 2014b)

The majority of seed production sources from natural populations although increasingly,

stocks are approaching or have exceeded maximum sustainable yields. Stock enhancement

through the capture of larvae while still relying on natural seed in both extensive and

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intensive forms of culture is a common practice worldwide. However the reliability of

natural recruitment can never be guaranteed, and conflicts over the use of the coastal zone

are becoming ever more pressing. One solution for meeting the growing demand for seed by

the mussel industry is seed production in hatcheries. However, production costs are still too

high in Europe compared to the wild-caught seed to turn it into a profitable business.

Hatchery production of spat could provide a back-up when natural spat fall fails, reduce the

pressure on the natural populations and eventually would allow for selection.

Mussels are farmed using long lines, rafts or a mixture of long lines and poles installed in

shallow intertidal areas. Aypa (1990) describes three main categories of culture methods for

mussel cultivation, bottom culture growing mussels directly on the bottom, intertidal and

shallow water culture in the intertidal zone, and deep water culture (Figure 2.8).

Figure 2. 8: Production cycle of Mytilus edulis (FAO, 2014b)

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The largest bivalve markets in the European Union (EU) are France, Italy, and Spain. France is

the largest EU importer of mussels (FAO, 2015). Meanwhile, smaller sized markets include

the Netherlands, Italy, Belgium and Denmark. Ireland and France provide the market with

oysters, while Denmark, Ireland, The Netherlands, and Spain provide mussels. The trade is

mainly intraregional between the EU Member States with a smaller contribution from third

countries. Only a few third countries, such as Chile and New Zealand, can penetrate the EU

markets with their mussel species.

2.3.5. Feeding behavior and digestive system of blue mussel

The blue mussel is a filter feeder, a feeding process that is well conserved amongst most

bivalve species. Food particles are taken up from the incoming water current, followed by

selection, transport, ingestion and finally digestion as described below (Jorgensen, 1990,

Gosling, 2003).

The water, with suspended organic particles such as unicellular phytoplankton, enters the

mussel through the inhalant opening. With the help of cilia, located on the gills, particles of

interest are trapped in mucus and transported along the labial palps towards the mouth. The

food particles are then, again assisted by cilia, transported through the esophagus and

directed towards the stomach. The stomach is surrounded by a dark digestive gland. Due to

mechanical and enzymatic processes, the food particles are digested extracellularly. These

digested end-products are then absorbed in the hemolymph (Figure 2.9).

Via the hemolymph this transport of nutrients could, for example, end up in the mantle

tissue where it is stored as the metabolic reserve glycogen. The waste materials and rejected

products are converted to faeces in the intestine and excreted through the anus. They find

their way back into the seawater through the exhalant opening (Jorgensen, 1990, Gosling,

2003). The selection of particles of interest is a selective process. The unwanted particles are

rejected and exit the mussel, as pseudofaeces, together with the waste water through the

inhalant opening (Jorgensen, 1996).

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Figure 2. 9. Digestive system of M. edulis: Illustration of the stomach and digestive gland

(Gosling, 2003)

Figure 2. 10: Schematic illustration of filter feeding process (Aquascope, 2000).

Bayne and Widdows (1978) determined the filtration rate of M. edulis to be 1.34-2.59 L h-1.

Winter (1973) found that filtration rate in mussels increases with body size. On average,

mussels with shell lengths of 8.5 mm filtered algal cells out of water at a rate of

approximately 20 ml h-1 whereas mussels with shell lengths of 56.5 mm filtered at a rate of

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about 1300 ml h-1. Winter (1973) also found that halving the algal concentration

approximately doubled the filtration rates. This large filter capacity allows the mussel to

ingest unselectively not only food particles and organic matter but also bacteria, toxic

substances, parasite larvae and even chemical pollutants. Because of this, the blue mussel

can concentrate several contaminants at high levels in its tissue and is thereby an ideal

biological marker for monitoring aquatic environments (Widdows et al., 2002, Brenner et al.,

2014). This characteristic also requires that mussels are produced in “clean” environments

and/or that they are regularly monitored for contaminants, including amongst others faecal

coliforms and marine toxin (depuration is often applied depending on the status of the

production area).

The life of marine bivalve mollusks is complex and includes a planktonic larval stage. Both

larvae and many adult bivalves feed by capturing microscopic particulates, inclusive detritus,

bacteria, microalgae and small animals (seston). Sestonic particles captured by bivalve

suspension feeders typically have diameters in the order of 1 to 100 μm: bacteria to

microplankton (Figure 2.11) and occur in seasonally variable amounts in seawater. The size

of particle captured depends on the different stage of life cycle of these species (Raby et al.,

1997)

Figure 2. 11: Comparative sizes of suspension-feeding bivalves and their potential food (seston). Adapted from Wildish and Kristmanson (1993).

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2.4. The immune system of bivalves

In common with most invertebrates, bivalve mollusks are characterized by an innate, non-

specific immune system. This means that they react towards foreign components without

any memory regarding previous contacts and they do not use antibodies, the latter being

elements of the adaptive immune system (Roch, 1999, Gosling, 2003). The innate immune

system in bivalves comprises both cellular and humoral components, which play a vital role

in protecting the animals against foreign invaders namely bacterial, fungal, and viral

pathogens (Canesi et al., 2002b, Iwanaga and Lee, 2005)

The cellular response of the innate immune system is based on the activity of the

hemocytes, which resemble the monocytes and macrophages of vertebrates in both

structure and function. The hemocytes, circulating in the hemolymph, are responsible for

different cell-mediated reactions such as phagocytosis and subsequent cytotoxic reactions

(Lee, 2005). Phagocytosis generally starts with the recognition of the foreign particle such as

a bacterium. This particle, engulfed by the phagosome, is further processed. Hemocytes

granules fuse with phagosome. This allows effector molecules to come into contact with the

foreign particles helping to destroy them. This effectors include the production of reactive

species of oxygen (ROS) through the NADPH oxidase complex and/or defense molecules

(antimicrobials, hydrolytic enzymes) (Bachère et al., 2015). These cellular components of the

immune system are also involved in other important physiological functions such as

digestion and nutrient transport, wound healing and shell excretion (Lee, 2005, Bettencourt

et al., 2009).

The humoral component immune system of bivalves consists of the blood cells, the

hemocytes, and of soluble Hemolymph factors, that operate in a co-ordinated way to

provide protection from invading micro-organisms (Canesi et al., 2006). Hemolymph serum

contains humoral defense factors, such as soluble lectins, hydrolytic enzymes and

antimicrobial peptides (Canesi et al., 2002a, Pruzzo et al., 2005, Bachère et al., 2015) (Figure

2.12). Bivalve hemocytes are extremely heterogeneous; classifications proposed for different

bivalve species are reported elsewhere (Fryer and Bayne, 1996, Ottaviani and Franceschi,

1998, Hine, 1999).

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Figure 2. 12: Mechanisms involved in the bactericidal activity of bivalve hemolymph (Canesi

et al., 2006)

2.4.1. Cellular immune response

Cellular response is carried out by hemocytes and in Mytilus edulis, they can be subdivided

into two categories, namely granulocytes and hyalinocytes or agranulocytes. Granulocytes

are the most abundant hemocytes in bivalve and have a large variability in number, type of

granules and degree of phagocytic activity. For example in Mytilus campechiensis, the

percentage of granulocytes and agranulocytes is 58.4 and 41.6, respectively (Huffman and

Tripp, 1982). The latter group, agranulocytes, has few or no granules and are less phagocytic

then granulocytes (Gosling, 2003, Song et al., 2010). The classification of hemocytes for

different bivalve mollusk species summarized in Table 2.2.

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Table 2. 2 Classification of the hemocytes for different bivalve

Bivalves species Hemocytes Reference

Clam Tapes philippinarum -Granulocytes (80.1%)

-Hyalinocytes (32.2%)

-Hemoblast (18.9%)

-Serous cells (0.8%)

Cima et al. (2000)

Giant Clam Tridacna crocea -Eosinophilic granular

-Hemocytes

-Agranular cells

-Morula-like cells

Nakayama et al. (1997)

Meretrix meretrix -Agranular hemocytes

-Lymphoid hemocytes

-Large granular hemocytes

-Small granular hemocytes

Yanyan et al. (2006)

Anodont (Anodonta cygnea) -Granulocytes (eosinophilic and

basophilic granulocytes and an

intermix)

-Agranulocytes (hyalinocytes and

blast-like cells, vesicular cells)

Salimi et al. (2009)

There are different types of cellular defense mechanisms in bivalves, namely hemocytosis,

phagocytosis and encapsulation.

Hemocytosis, or hemocyte proliferation, is the first response to an infection or presence of a

non-self-particle. This involves an increase of circulating hemocytes, which move towards

the infected of injured tissue (Gosling, 2003). This movement of the cells is activated by

chemo-attractant substances, such as opsonines (for example, the extrapallial protein in

Mytilus galloprovincialis (MgEP)), to which the hemocytes are sensitive to. (basically

opsonization of microorganisms has been shown to occur through plasma proteins like Cg-

EcSOD, which promotes b-integrin-mediated phagocytosis (Duperthuy et al., 2011)). This

movement is implemented by two motile responses, namely chemotaxis and chemokinesis.

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Chemotaxis is a directional movement and in M. edulis for example, is triggered by the

presence of lipopolysaccharides from Escherichia coli and Serratia marcescens. Chemokinesis

is a random, indirectional migration of the hemocytes and for example stimulated by N-

formyl-methionyl-leucyl-phenylal-anine (N-FMLP) in the blue mussel (Canesi et al., 2002b,

Song et al., 2010).

Myticin class C of mussel Mytilus galloprovincialis (Myt C) is known as an antimicrobial

peptide, and it is the most abundantly expressed gene in cDNA after immune stimulation of

Mytilus galloprovincialis (Costa et al., 2009). Recently, Balseiro et al. (2011) showed that this

AMP (Myt C) could be acting as immune system modulator molecule because its over-

expression was able to alter the expression of mussel immune-related genes such as Myticin

B, Mytilin B, and lysozyme. Moreover, the in vitro results indicate that Myt C-peptides have

antimicrobial and chemotactic properties. The result of this study suggested that Myt C

should be considered not only as an AMP but also as the first chemokine/cytokine-like

molecule identified in bivalves.

After this movement, non-self-recognition and attachment of the hemocyte to the targeted

particle occurs and, the particle is enclosed in a vesicle in the cell by endocytosis. This vesicle

is also called a primary phagosome and fuses together with a lysosome to form a

phagolysosome. In this structure, the engulfed particle is destroyed by lysosomal enzymes,

reactive oxygen intermediates, nitric oxide and antimicrobial factors (Gosling, 2003, Song et

al., 2010). Antimicrobial factors including cytoskeleton modification, internalization, and

destruction of the engulfed target within phagosomes (Song et al., 2010). This mechanism is

called phagocytosis and is illustrated in Figure 2.13.

Nodulation is one of cellular defense mechanisms against bacterial, fungal and viral infection

in insect (Drosophila) and other invertebrates. Nodulation is mediated by hemocytes

aggregate formation around bacteria and fungi (Lavine and Strand, 2002). Encapsulation is

similar to nodulation and refers to hemocyte aggregation around larger pathogens like

parasitoids and nematodes (Gandhe et al., 2007). The nodulation mechanism has been

exited in bivalve Eastern oyster Crassostrea virginica. However, the study of nodule

formation in bivalves is very rare.

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Figure 2. 13: Schematic illustration of phagocytosis (Anonymous, 2016).

Encapsulation is the cellular immune response to foreign particles that are too large to be

phagocytosed. In this mechanism the foreign particle is encapsulated with hemocytes and

cytotoxic products, such as degradative enzymes and free radicals, are released to destroy

the particles (Song et al., 2010).

2.4.2. Humoral immune defense

Humoral response is carried out by humoral defense factors that are present in the

Hemolymph, as mentioned above. These factors can cause a direct cytotoxic effect on

pathogens and a lot of soluble molecules in the hemolymph can be categorized as humoral

defense factors. It should be noted that some of these factors are present in hemocytes and

are thereby also involved in the cellular defense. The most abundant humoral defense

factors in Mytilus species are (soluble) lectins, hydrolytic and lysosomal enzymes such as

phosphatase and lysozyme and antimicrobial peptides (Canesi et al., 2002b, Pruzzo et al.,

2005).

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2.4.2.1. Lectins

Lectins are sugar-binding proteins that can interact specifically and reversibly with

membrane glycoproteins or glycolipids of bacteria or other invaders (Renwrantz, 1990).

Thereby, lectins are involved in several immune functions, such as self/non-self recognition

and associated effector mechanisms (Song et al., 2010).

Lectins can have a direct or indirect role in the phagocytosis process. Lectins can cause

agglutination and thereby potentiate the phagocytosis process by immobilization of

bacteria. On the other hand, lectins can also act as bridge molecules between bacteria and

hemocytes and thereby act as opsonization for phagocytosis (Pruzzo et al., 2005). There exist

many lectins, and they can be distinguished from each other by differences in molecular size,

subunit structure, agglutination properties, and sugar-binding specificity (Canesi et al.,

2002b). There are some important lectins such as MytiLec which is a α-Gal-binding lactic that

was isolated from the Mediterranean mussel Mytilus galloprovincialis (family Mytilidae)

(Hasan et al., 2015) and C-type GalNAc-specific lectins recovered from the sea cucumber

Cucumaria echinata which interact with target membranes and exhibit strong hemolytic

activity and cytotoxicity through pore formation (Hatakeyama et al., 1996).

2.4.2.2. Lysozymes

Lysozyme is a widely distributed anti-bacterial molecule (basically mostly a family of

molecules) present in numerous animals including bivalves. It is a hydrolytic protein that can

break the glycosidic union of the peptidoglycans of the bacteria cell wall. It is present in the

Hemolymph and the cells, and its release is associated with physiological changes, age,

stress, (Lopez et al., 1997, Cronin et al., 2001) and bacterial infections (Li et al., 2008).

Several studies have shown that lysozymes are able to kill Gram-negative bacteria, but some

have a wider activity. It was demonstrated for example that: an antibacterial lysozyme-like

protein from Icelandic scallops could inhibit the growth of all Gram-positive and Gram-

negative bacteria. Moreover, purified oyster plasma lysozyme inhibited the growth of Gram-

positive bacteria (e.g., Lactococcus garvieae, Enterococcus sp.) and Gram-negative bacteria

(e.g., Escherichia coli, Vibrio vulnificus)(Yue et al., 2011).

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Lysozyme expression has been detected and localized in cells of digestive tubules, gill and

mantle (Bachère et al., 2015). Although lysozyme activity was first reported in mollusks over

30 years ago, complete sequences were published only recently, including those from M.

edulis, M. galloprovincialis, and two different forms in Crassostrea virginica, C. gigas, Ostrea

edulis and Chlamys farreri (Bachali et al., 2002, Itoh and Takahashi, 2007, Li et al., 2008).

Several types of lysozymes have been purified, from the best-known chicken-type (c-type)

and goose-type (g-type) to the more recently identified invertebrate-type (i-type)(Nilsen and

Myrnes, 2001). Such i-type lysozymes have been identified in several bivalve mollusk,

including the Icelandic scallop, Chlamys islandica (Nilsen and Myrnes, 2001) and the blue

mussel, Mytilus edulis. In the Mediterranean mussel, M. galloprovincialis, lysozyme has been

found localized within granular hemocytes. Higher levels of activity have been detected in

hemocytes compared with plasma in both M. edulis and the carpet shell clam, Ruditapes

decussatus (Li et al., 2008).

2.4.2.3. Antimicrobial peptides (AMPs):

AMPs are a major component of the innate immune defense system of bivalves. AMPs are

molecules with a mass less than 10 kDa which shows antimicrobial and antifungal properties

(Tincu and Taylor, 2004). Currently, ca. 10 different types of AMPs have been identified in

the mussel. They can be categorized into four different groups according to their

antimicrobial and antifungal action and to their primary structure (hydrophobic, cationic or

amphipathic). These four groups with their associated classes are called defensins, mytilins,

myticins and mytimycins (Canesi et al., 2002b, Tincu and Taylor, 2004, Song et al., 2010).

Many antimicrobial peptides are located in epithelia (Schröder, 1999) where they prevent

invasion by pathogens while others may be especially abundant in circulating cells.

In bivalves, AMPs were first purified from the blue mussel (M. edulis) hemocyte granules

(Charlet et al., 1996) and classified into three families: defensins, related to arthropod

defensins, mytilins and mytimycin. These three AMPs are present in different quantities

(Gestal et al., 2008). Moreover, the genes are differentially regulated according to the

challenging bacteria (Cellura et al., 2007). In M. galloprovincialis, all of the peptides possess

eight cysteine’s arranged in particular conserved arrays (Mitta et al., 2000a, Mitta et al.,

2000d). The typical structure of these peptides is shared by the new myticin class, myticin C,

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recently described in M. galloprovincialis (Pallavicini et al., 2008). The genes encoding these

proteins showed a high polymorphic variability (Padhi and Verghese, 2008), which has been

suggested to account for the high resistance of the Mediterranean mussel to disease (Costa

et al., 2008). This variability was also observed in clam and mussel mytilins (Gestal et al.,

2007, Parisi et al., 2009). In the oyster C. gigas, a molecular biological approach revealed the

presence of two isoforms of a defensin-like protein (Gueguen et al., 2006, Gonzalez et al.,

2007). In contrast to the one from the mussel, the oyster defensin was not altered after a

bacterial challenge (Gueguen et al., 2006).

Table 2. 3: Antimicrobial peptides of two closely related mussel species and their classes

(Tincu and Taylor, 2004)

Species Peptide Class

Mytilus edulis Defensins A and B

Mytilin A and B

Mytimycin

β-3

β-4

β-6

Mytilus galloprovinciallis Myticin A and B

Defensins 1 and 2

MytilinB, C, D and G1

β-4

β-4

β-4

2.4.2.4. Phenoloxidase (PO)

Phenoloxidase is a critical component of the immune system of mollusks (Asokan et al.,

1997, Lopez et al., 1997). As the product of a complex cascade of reactions, PO is generated

from prophenoloxidase (proPO) through a limited proteolysis by a proPO activating enzyme

(ppA) (Coles and Pipe, 1994). In vivo ppA is activated in the presence of microbial

polysaccharides, such as lipopolysaccharides, peptidoglycans and 1,3-β-glucans. Upon

binding of these type of molecules to specific pattern-recognition receptors ppA is activated,

and as a consequence, active ppA converts proPO to PO (Aspan et al., 1995, Ballarin et al.,

1998). Finally, PO is involved in melanization, encapsulation, wound healing, phagocytosis,

and pathogen extermination (Bai et al., 1997, Newton et al., 2004, Munoz et al., 2006). In

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bivalve mollusks, PO is mainly found in the Hemolymph (Asokan et al., 1997, Munoz et al.,

2006), and exits both in the soluble or cellular form: the soluble form is always involved in

humoral immunity, while the cellular PO, which binds to the surface of hemocytes is more

associated with cell-mediated immunity (Ling and Yu, 2005, Hellio et al., 2007), although

there is other PO-like activity (Rizzi et al., 1994). Significant differences in phenoloxidase

activity have been observed in bivalves exposed to environmental stressors or pollution

(Kuchel et al., 2010, Luna-Acosta et al., 2010). Phenoloxidase enzymes can be detected in all

life stages of M. edulis (Coles and Pipe, 1994, Dyrynda et al., 1995, Luna-Acosta et al., 2011)

and Pacific oyster C. gigas (Thomas-Guyon et al., 2009). PO enzymes are most commonly

found in adult hemocytes (Luna-González et al., 2003) but embryonic cells, which possess

some phagocytic capacity, possess a restricted level of PO activity as well (Dyrynda et al.,

1995, Luna-González et al., 2003) and it has been suggested that it plays an important

function in non-self-recognition and host immune reactions in the early life stages of bivalve

(Thomas-Guyon et al., 2009).

2.4.2.5. Production of reactive oxygen species (ROS)

Reactive oxygen (ROS) and nitrogen species (RNS) are naturally produced in all cells and

organisms. ROS generation may result in oxidative stress (Donaghy et al., 2015). In

mammals, all the blood phagocytes contain granules rich in lysosomal enzyme, which

degranule during phagocytosis to bring about the killing an degradation of endocytosis

pathogens (Pipe, 1992). In bivalve mollusks, ROS is produced in hemocytes as part of their

basal metabolism as well as in response to endogenous and exogenous stimuli (Donaghy et

al., 2015). The major reaction cascades from the initial pathogen contact through oxidative

killing are summarized in Figure 2.14.

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Figure 2. 14: Diagram of the major reaction cascades from the initial pathogen contact

through oxidative killing. Adapted from Roch (1999).

2.4.2.6. Cytotoxic enzymes activity

In many invertebrate species, several kinds of immune-related humoral activities have been

reported. Bivalves possess various types of non-specific humoral defense molecules

including agglutinins, opsonizing lectins, bactericidins, lysozymes and serine proteases. In the

blue mussel, M. edulis, a modification of the in vitro plaque assay has been employed to

demonstrate the presence of cytolytic molecules (another name is cytotoxic hemocytes

(Wittke and Renwrantz, 1984, Leippe and Renwrantz, 1988). Also, the plasma of the

Mediterranean mussel, M. galloprovincialis, contains cytotoxic activity against both

vertebrate (erythrocytes and mouse tumor) and protozoan cells. The prokaryotic bacteria,

Escherichia coli and Vibrio alginolyticus, were not sensitive to this cytotoxicity (Hubert et al.,

1996). Injection of erythrocytes stimulated the cytotoxic activity of the plasma with a

maximum 2 days post-injection, suggesting that cytotoxic molecules are involved in immune

defense (Hubert et al., 1997). The activity was still present in dialyzed samples but was

destroyed by heating at 45°C. Purification by anion exchange chromatography followed by

gel filtration revealed a 320-kDa cytotoxic polymeric protein (Roch et al., 1996). Composed

of three different proteins (25 – 320 kDa), the complex acts through a hetero-polymerization

process after binding to target cell membranes as revealed by ultrastructural observation

(Hubert et al., 1997).

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2.4.3. Difference in the immune system between bivalve adult and larvae

Very little is known about the immune system of bivalve mollusk larvae although some

studies reported that larvae of certain groups of mollusks may have a stronger innate

internal defense than others, or perhaps there are differences in the maturation rate of the

immune system during the early development (Luna-Gonzalez et al., 2002).

Dyrynda et al. (1995) found that larvae have non-specific defense barriers to bacterial

invasion as well as specific immune functions. Non-specific immune defense includes

external and mucosal barriers and some adaptive components that are transferred from the

mother, such as agglutinins, precipitins, lysins and immunoglobins on the egg membrane

surface (Mulero et al., 2007). External and mucosal barriers are the first line of defense since

infection usually requires intimate association with larvae surfaces (Olafsen, 2001, Gomez-

Leon et al., 2008).

In a study of the immune system of larvae of blue mussel M. edulis Dyrynda et al. (1995)

demonstrated some immune defense functions including production of degradative

enzymes phenoloxidase and arylsulphatase, phagocytosis of E. coli cells and generation of

reactive oxygen metabolites. Immunity attributes were compared to those of adults in Table

2.4 (Dyrynda et al., 1995), which indicates that some elements of the immune system in

adults bivalves also appear in the trochophore and veliger larvae.

Recently, Song et al. (2016) reported that the newly hatched, trochophore stages of the

Pacific oyster are not only critical for oyster development in general, but also critical for the

ontogenesis of the immune system. Phagocytosis was firstly observed in the early D-veliger

larvae, 17 hour after fertilization

The adult bivalve immune system does not appear to operate autonomously in regulation

and action as previously thought, but rather may interact with the nervous and endocrine

systems (Koller, 1990). These links may increase the complexity of the immune response and

potentially make it more sensitive to environmental stressors (Parry and Pipe, 2004).

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Table 2. 4: Summary of defense mechanism recorded in blue mussel (Mytilus edulis)

Activity Adults Larvae Reference

Degradative enzyme + + (Pipe, 1990)

Phagocytosis + + (Noël et al., 1994)

Cytotoxic reaction + ND* (Leippe and Renwrantz, 1988)

Reaction oxygen metabolites generation + ND (Pipe, 1992)

Nitric oxide generation + ND (Ottaviani et al., 1990)

(*)ND: not determined

2.5. Constraints of bivalve aquaculture

One of the main problems in the aquaculture of bivalve mollusks is occurring mortality,

which severely reduces production. These outbreaks of disease affect larval, and postlarvae

in the hatchery, as well as juvenile and adults, cultured in the natural environment. Research

has been conducted to determine the aetiology, epidemiology and control measures for

these epizootics (Renault, 1996). Bacteria, viruses, fungi, and protozoan parasites have

caused major epizootics in bivalve mollusks. This is probably facilitated by considerable

(commercial) movement of life animals, explaining the appearance and the spread of some

infectious diseases in several countries around the world (Renault, 1996). In this chapter, we

will focus on bacteria and viral diseases of bivalve mollusks that have been reported so far,

and their economic impact on the bivalve mollusk aquaculture.

2.5.1. Bacterial diseases

For the main microbial diseases affecting cultured marine bivalves the aetiologic agents have

been characterized, and their pathogenesis and pathogenicity have been studied. Several

recent bivalve-interaction models have been studied for the brown ring disease, juvenile

oyster disease, Pacific oyster nocardiosis and summer mortalities of oysters (Paillard et al.,

2004). In addition, the taxonomy and phylogeny of new potential bivalve pathogens and

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their virulence factors have been established. Facing the difficulty of identifying bacterial

strains associated with molluscan diseases (mainly vibriosis), polyphasic approaches have

been developed to correlate the phenotype and genotype of potential pathogens (Paillard et

al., 2004).

So far, in bivalve mollusk hatcheries, massive mortalities can result in the complete loss of

the production stock, with serious economic consequences. In most cases, the studies have

demonstrated that the problems are caused by bacterial pathogens, the primary etiological

agents being members of genus Vibrio, Pseudomonas, Aeromonas, and Vibrio–like bacteria

(VLB) (Tubiash et al., 1965, Tubiash et al., 1970, Sugumar, 1998, Beaz‐Hidalgo et al., 2010).

Although all life larval stages are vulnerable, the larvae are particularly exposed to a high

concentration of potentially pathogenic bacteria associated with tank surface, moribund

larvae, or organic detritus (moribund larvae or organic debris) during the temporary fixation

of the larvae on the bottom of the tank (Sutton and Garrick, 1993).

Vibrio alginolyticus has been reported as one of the most pathogenic bacteria for molluscan

larvae (Tubiash and Otto, 1986, Luna-Gonzalez et al., 2002). For example, survival rates of

Mytilus galloprovincialis larvae exposed to Vibrio alginolyticus were found to be between 64-

95% 48 h post challenge for the different bacterial concentrations. Minimum survival (64%)

was found in 3 × 104 CFU ml–1 and the maximum in the control treatment (95%) (Anguiano-

Beltrán et al., 2004).

Together with the species V. alginolyticus, both V. tubiashii, and V. anguillarum were

recognized as the primary causal agents of the “bacillary necrosis” (Tubiash et al., 1970,

Tubiash and Otto, 1986). The disease is characterized by bacterial colonization of the mantle,

velum disruption, abnormal swimming, visceral atrophy, and lesions in the organs among

other signs. Another characteristic sign of larval vibriosis in hatcheries is the appearance of

the phenomenon called “spotting,” defined as an accumulation of moribund larvae

agglutinated at the bottom of the tanks (DiSalvo et al., 1978). Table 2.5 provides a summary

of the most recent studies on bivalve larvae vibriosis.

Vibriosis is not only the main cause of massive mortalities in larval stages but also effects

juveniles and adults (Paillard et al., 2004). A total of 90 samples of adults blue mussels grown

in Germany were analyzed. The analysis revealed the presence of Escherichia coli and

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Salmonella spp. but also may Vibrio spp. Vibrio spp. was detected in 74.4% of the samples

analyzed in this study. Among Vibrio isolates, Vibrio alginolyticus was the species most

frequently detected (51.2%), followed by Vibrio parahaemolyticus (39.5%) and Vibrio

vulnificus (3.5%). V. parahaemolyticus and V. vulnificus were not found in samples collected

at low water temperatures (Lhafi and Kuhne, 2007).

In France, classical Vibrio infections are associated with the phenomenon known as Summer

Mortality (SM) in juvenile Pacific oysters (Crassostrea gigas). SM affects juvenile oyster

populations during the warmer months when the water temperature is ≥ 18 0C and

reproduction takes place (Berthelin et al., 2000). This phenomenon has been associated with

stress situations, low dissolved oxygen or presence of toxic substances in the sediment

(Cheyney et al., 2000). Lipp et al. (1976) were the first to observe that the oyster

Hemolymph had high levels of Vibrios that were causing death and later studies identified V.

splendidus as the causal agent of SM (Lacoste et al., 2001, Gay et al., 2004). More recently,

Allain et al. (2009) suggested the possible role of V. harveyi as the aetiological agent of SM,

since it was detected in most samples of affected oysters during the 2008 warm season and

was able to produce mortality in experimental challenges. The most accepted theory today

is that the oyster SM cannot be attributed to one bacterial pathogen or to the Oyster

Herpesvirus alone, but to a complex interaction between the physiological and/or

genetic state of the host, environmental factors and the presence of various opportunistic

infectious Vibrio species (Paillard et al., 2004, Pruzzo et al., 2005, Labreuche et al., 2006).

Another serious mollusk disease is the Brown Ring Disease (BRD), caused by V. tapetis

(Borrego et al., 1996). It has been widely studied since it is the primary disease with bacterial

etiology in adult clams (Ruditapes semidecussatus and Ruditapes decussatus) (Paillard et al.,

2004). Susceptibility to V. tapetis infections is species specific, causing greater physiological

disturbances and mortality in R. semidecussatus than in other species of clams (R.

decussatus and Mercenaria mercenaria) or in the oyster C. Virginia (Allam et al., 2001, Allam

et al., 2006). Environmental factors (i.e. temperature and salinity) play a role in the

development of BRD, which tends to be more frequent in the spring and winter as the

optimum growth temperature for V. tapetis is 15 °C (Paillard et al., 1994, Paillard et al.,

2004).

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Table 2. 5: Experimental infection of bivalve larvae, juvenile and adults with pathogenic Vibrio spp. Adapted from Romalde and Barja (2010).

Pathogenic species Bivalve species* Life stage Reference

V. alginolyticus M.galloprovincialis Larvae Anguiano-Beltrán et al. (2004)

R. decussates Larvae Gómez-León et al. (2005)

V. splendidus biovar II

R. decussates Larvae Gómez-León et al. (2005)

C. gigas Juveniles Waechter et al. (2002)

V. splendidus –like

P. maxima Larvae Sandlund et al. (2006)

C. gigas Adults Gay et al. (2004)

V. aestuarianus C. gigas Adults Garnier et al. (2008)

V. tubiashii C. gigas Larvae Hasegawa et al. (2008)

V. parahaemolyticus

V. vulnificus

V. alginolyticus

Mytilus edulis Adults Lhafi and Kuhne (2007)

Vibrio sp.

C.gigas; O. edulis Larvae Estes et al. (2004)

C. virginica Larvae Gomez-Leon et al. (2008)

C. gigas; C. virginica Juveniles Gay et al. (2004); Gomez-Leon et al. (2008)

*Abbreviations: C = Crassostrea, M = Mytilus, O = Ostrea, P= Pecten, R = Ruditapes.

2.5.2. Viral diseases

In general, viruses can be significant infectious agents in bivalve species. Particularly in early

life stages of bivalves, there appears to be mortality associated with Herpes-like and irido-

like viruses (Elston, 1997). The herpes-like virus affects bivalve populations in many countries

including Australia, Asian countries and especially in France where infected hatchery-reared

larval Pacific oysters showed cumulative infection rates of 60-100% at 7-11 days post

fertilization (Hine et al., 1992). Larval hatchery-reared Pacific oysters in France experienced

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abnormal mortality and morbidity associated with a herpes-like viral infection (Nicolas et al.,

1992) and mass mortality of 80-90% observed during the summer. Le Deuff et al. (1994)

reported experimental transmission of the infection from a population of infected oyster

larvae to larvae obtained from a pathogen-free broodstock but maintained by axenic

methods. Within 48 h of inoculating a virus suspension into the cultures of axenic larvae, the

animals exhibited a high rate of morbidity, and virus-like particles were observed in

connective tissue cells around the velum. Le Deuff et al. (1994) later showed that the viral

infection would produce complete viral particles and 80-90% mortality in oyster larvae

reared at 25-26 °C but not in larvae reared at 22-23 °C. At the lower temperature, the

authors reported nuclear alterations but no viral particles were found. This could be

interpreted as latent infections. Elston (1997) also found viral infections developing in larvae

from three out of four broodstock batches obtained from different areas in Atlantic France.

Larval hatchery-reared Pacific oysters in France experienced unusually high mortality and

morbidity rates associated with a herpes-like viral infection (Nicolas et al., 1992). The

infection was associated with cellular changes in oyster seed, collected from survivors of

populations that had undergone 80-90% mortality during the summer for at least 20 years

(Renault et al., 1994, Goulletquer et al., 1998, Soletchnik et al., 2007). This syndrome has

been reported in most Pacific oyster producing countries, such as Japan, USA and Australia.

The first description of this phenomenon was made in the 1940s in Japan, where C. gigas is

endemic. Herpes virus also causes mortality in different bivalve aquaculture species. For

example, abnormal summer mortalities associated with the detection of ostreid herpesvirus

1 (OsHV-1) have been reported among larvae and spat of the Pacific oyster C. gigas in France

(Schikorski et al., 2011), as well as in farmed seed and adult green-lipped mussels (Perna

canaliculus) from New Zealand. Cumulative mortality of 50-100% associated with apparent

virus infection was reported in green lipped mussel seed during the summer and autumn,

while mortality in adults was only 2-5% (Jones et al. (1996). In addition, summer mortality

occurred in different mollusks species and regions such as the blue mussel (M. edulis) in

Denmark (Rasmussen, 1986), abalone (Haliotic spp.) farm in Australia (Hooper et al., 2007,

Tan et al., 2008, Corbeil et al., 2012).

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Among 1200 specimens of M. edulis examined from three localities in Denmark, a total of 34

mussels with virus-associated granulocytomas were discovered. Confirmation that the virus

was related to Picornaviridae was based on the following five observations. The virion was

non-enveloped, the capsid was apparently icosahedral and measured approximately 27 nm,

the virus was found only in the cytoplasm, and no viral DNA was seen (for the latter the

Feulgen reaction was used for viral DNA detection. However, result of the Feulgen reaction

was negative) (Rasmussen et al., 1985).

2.6. Solutions to control diseases in bivalve larvae culture

2.6.1. Water treatment

The influence of the environment on hatchery cultures is paramount. The bivalve larvae live

in the water, sharing the environment with both beneficial bacteria and potential pathogens.

The bacterial loads in bivalve larvae tanks and reported to vary between 105- 106 bacteria

ml-1 (Nevejan et al., 2016). As bivalves release their young at an early ontogenic stage, these

early life-history stages are especially sensitive to possible infections. In part, this is due to

their filter-feeding behavior and the subsequent flow of sea water through the organism

(Prado et al., 2010). Some methodologies and technologies are currently used to remove the

excess of organic matters and nutrients in aquatic systems. Different water treatments, such

as filtration, pasteurization, Ozone, Ultraviolet (UV) radiation have been employed in

mollusk hatcheries (Vasconcelos and Lee, 1972, Murchelano et al., 1975, Lodeiros et al.,

1987). A recent alternative approach is the integration of a biological filter in recirculating

systems (Marinho-Soriano et al., 2011) The core mechanism of such strategies is the

utilization of living bacteria and algae to convert the toxic pollutants into the less toxic form

or microbial biomass that can be fed to the culture animals.

Once most of the solid particles are eliminated from the seawater through decantation, the

water is filtered. Filtration is an expensive treatment, with incremental costs with increased

volume and the filtration range established. An advisable practice is to reduce the content

of bacterial load and results show that this is better than using alternative systems like

pasteurization, as demonstrated by Lewis et al. (1988) in a Pacific oyster hatchery. The main

disadvantage of pasteurization is the risk of subsequent contaminations due to the necessity

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of transporting the treated water through long pipes and the prolonged period of high

temperature to cool down (Prado et al., 2010).

To overcome those problems, disinfection with chlorine has been employed too, but it

comes with many problems, like the interference with the larval mechanism of pumping, as

described by Vasconcelos and Lee (1972) for cultures of C. gigas, and by Asokan et al. (2013)

for a mussel hatchery. Some studies suggested that the reactions between chlorine and

organic nitrogen in the water could produce residues toxic to marine organisms (Jorquera et

al., 2002). Another alternative is ozonization, but its application to disinfect aquaculture

systems can be complex and costly (Summerfelt, 2003). One of the most common

treatments is the radiation of seawater with ultraviolet (UV) light, with unquestionable lethal

effects on bacteria and viruses. However, there are disagreements about the actual effects

when the water treatment is used in hatcheries. Vasconcelos and Lee (1972) found

advantages in its use, as different bacterial populations, including bivalve pathogens,

decreased. In larval cultures of Ostrea edulis, the population of Vibrios decreased with UV

treatment (Lodeiros et al., 1987, Sainz-Hernandez and Maeda-Martinez, 2005).

Nevertheless, Brown (1981) found that the same treatment with UV tested against two

pathogenic Vibrio spp. was effective against one of them, while the other strain was able to

grow after an initial inhibition. Therefore, the effects of UV-radiation are not homogenous

against all the bacterial populations, and more variability is added with factors like the dose,

the water flow and the individual efficiency of the radiation unit (Prado et al., 2010). In

general, those treatments can reduce number of bacteria in water. For example the addition

of ozone reduced the numbers of heterotrophic bacteria by 3log10 units (Suantika et al.,

2001). A reduction in bacterial numbers can only have advantages in terms of the hygienic

quality of aquatic animal; however, attention should also be paid to the changes in bacterial

communities imposed by the disinfection.

Moreover, it is known that the efficiency of the treatment is affected by the organic content

of water and the presence of small particles to which bacteria are attached (Liltved and

Cripps, 1999). In summary, these significant differences in the effectivity can lead to the

selection of undesirable populations, including those resistant to the treatment, which

would find an ecological niche favorable to their growth.

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Currently many bivalve mollusk hatcheries are using a flow through system (FTS) for growing

larvae. There are some important advantages larval handling is reduce and the water

parameters are less fluctuating because the feeding with algae happens also continuously.

These stress-reducing conditions make the larvae less susceptible for disease. Besides a

better disease control, larval tanks can hold a much higher larval density in comparison to

batch culture and labor cost are reduced (Magnesen and Jacobsen, 2012). However, one

should take into account that it is impossible to create a stable bacteria community in the

tanks under these conditions and the development of bivalve- specific recirculating systems

offers a window of opportunities.

2.6.2. Antibiotics

Antibiotics have been extensively used as therapeutic treatments to disease in bivalve

hatcheries since Davis and Chanley (1956) showed their ability to reduce larval mortalities in

hard clam Mercenaria mercenaria. However, in parallel with studies confirming the

beneficial effects, other works described toxic effects, as well as a lack of effectiveness or

consistency in the results obtained. Jeffries (1982) observed that the treatment with

chloramphenicol achieved a slight recovery in a larval culture of C. gigas, but at the same

time the larvae ceased swimming and therefore feeding. Uriarte et al. (2001) reported that

chloramphenicol added to the food two times per day resulted in better survival and growth

rates in Chilean scallops (Argopecten purpuratus) between the early larvae and pediveliger

stages cultured in a closed system. Similar benefits were reported for larval stages of the

great scallop (Pecten maximus) (Torkildsen et al., 2005). In experiments with the blue

mussel, M. edulis, the treatment with chloramphenicol resulted in good survival but slow

development, showing an opposite effect to the combination of ampicillin and streptomycin,

with excellent development but low survival (Prado et al., 2010).

However, the massive use of antibiotics has led to the development of multiple drug

resistant bacteria (Karunasagar et al., 1994, Tendencia and de la Peña, 2001) and even the

risk of transmission of resistant plasmids among aquatic-associated pathogens to human

pathogens, with the subsequent risk for the hatchery workers (Jeffries, 1982, McPhearson et

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al., 1991, Spanggaard et al., 1993). Also, the water exchange between the hatchery and the

environment may spread resistant bacteria (McPhearson et al., 1991, Kemper, 2008).

As a result of the growing awareness that antibiotics should be used with more care, more

strict regulations on antibiotic usage in aquaculture and on the acceptable residue levels of

various agents in the aquatic product have been set (Bachère, 2003, Defoirdt et al., 2011).

For instant, nowadays, chloramphenicol is prohibited in any veterinary applications to avoid

the health hazards to humans by food consumption at least within the EU (Annex IV of

Directive 2377/90/EEC), due to its potential toxic effects in humans as well as to the

difficulty in determining security levels of its residues (Prado et al., 2010).

The final objective of all the methods cited above is to obtain a substantial reduction of

number bacteria within the hatcheries, but this goal does not seem reasonable, since the

bacteria population, or, at least, part of it, may have a beneficial effect on larval

development. In addition, a lack of microbiota can favor the colonization of the system by

non-desirable microorganisms, due to a lack of natural competitors, in an environment with

a regular input of organic matter and optimal conditions (Romalde and Barja, 2010).

2.6.3. Immunostimulants

A set of different substances such as beta-glucans, bacterial products, and plant constituents

may directly activate the innate defense mechanisms by acting on receptors. This may

trigger the production of anti-microbial molecules. These immunostimulants are often

obtained from bacterial sources, brown or red algae. Terrestrial fungi are also exploited as a

source of novel potential substances. The use of immunostimulants, as dietary supplements,

can improve the innate defense of animals providing resistance to pathogens during periods

of high stress (Bricknell and Dalmo, 2005).

Heat Shock Protein (HSP) was considered as an immunostimulant in different bivalve mollusk

species, including the mud clam Mya arenaria (Abele et al., 2002) and Pacific oyster C.gigas

(Lang et al., 2009). These studies found that transcription after heat shock increased for

genes putatively encoding heat shock proteins and genes for proteins that synthesize lipids,

whereas transcription decreased for genes of proteins that mobilize lipids and detoxify

reactive oxygen species. Hong et al. (2006) reported that the genomic DNA of Escherichia

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coli, which contains the un-methylated CpG motif, was used to evaluate the

immunostimulating effect of bacterial DNA on innate immune responses in the bivalve

mussel Hyriopsis cumingii Lea. These results showed that the bactericidal activity of the

hemocytes was significantly increased when the cells were incubated with 50 or 100 μg ml-1

bacterial DNA for 12 and 24 h. Antibacterial activity, lysozyme activity, and prophenoloxidase

(proPO) production of hemolymph were also increased, when the bivalve mollusk was

injected with 50 or 100 μg ml-1 of bacterial DNA for 12 and 24 h. These activities returned to

the control level after 48 h. In summary, bacterial DNA with unmethylated CpG motif

(Unmethylated CpG motifs are prevalent in bacterial but not vertebrate genomic DNAs)

could activate some parameters of the immune system of bivalve mollusk both in vivo and in

vitro.

In conclusion, immunostimulants can reduce the losses caused by disease in aquaculture;

however, they may not be effective against all diseases. For the efficient use of

immunostimulants, the timing, dosages, method of administration and the physiological

condition of aquatic animal, including bivalve mollusks, need to be taken into consideration.

2.6.4. Prebiotics and probiotics

Since the concept of prebiotics was introduced, it has attracted scientific and commercial

interest. The main characteristic of prebiotics is the selective stimulation of intestinal

bacteria growth, contributing to the hosts' health and welfare. This requires prebiotics to be

non-digestible and resistant to gastric acidity, bile salts, and hydrolysis by the host's enzymes

(Glenn and Roberfroid, 1995, Roberfroid, 2007). Symbiotics, products that contain both

probiotics and specific prebiotics that promote the growth of the probiotic strains, are

considered a good solution in many cases (Apajalahti et al., 2004, Cheng et al., 2005, Bomba

et al., 2006). Recently, the use of prebiotics has become popular for a number of

aquaculture species, both fish and shrimp (Yousefian and Amiri, 2009). Some of the more

commonly used prebiotics in animal aquaculture feeds include inulin, fructooligosaccharides

(FOS) and transgalactooligosaccharides (TOS) (Vulevic et al., 2004), which function to

increase feed intake, growth rate and food digestibility of aquatic animals (RingØ et al.,

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2010). In bivalve mollusk species, the use of prebiotics for larval stages has not been as

widely used compared to probiotics.

Verschuere et al. (2000) gave a definition of probiotics, appropriated to be used within the

field of aquaculture, taking into account the close interaction of the animal with the

environment. According to these authors a probiotic would be a live microbial additive with

a beneficial effect on the host, modifying the microbiota associated with the host or

the environment, ensuring an optimal use of the feed or improving its nutritional value,

improving the host response to the disease, or getting a better quality of its environment.

To date, the use of probiotics in aquaculture has focused mainly on fish and crustacean

production, with scarce literature considering their application in bivalve mollusk

aquaculture. Lodeiros et al. (1989) published the first work on the effect of antibiotic-

producing marine bacteria on the larval survival of scallop Pecten ziczac and the best results

were achieved using a live bacterial culture.

Douillet and Langdon (1993) conducted a set of experiments with axenic oyster larvae (C.

gigas), fed with a mono-algal axenic diet and inoculated with different marine bacteria. Most

of the isolates assayed had negative or neutral variable effects, and only the strain CA2

(Alteromonas sp.) showed a beneficial impact on the growth and survival of oyster larvae. In

non-axenic larval cultures, Douillet and Langdon (1994) found that the effect of strain CA2

depended on its concentration, being detrimental to larval growth and survival when

administered at 107 CFU ml-1 whereas at 10 to 106 CFU ml-1 larval survival was not affected

and growth was enhanced. The optimal concentration, 105 CFU ml-1, was within the constant

values for bacterial populations in bivalve cultures obtained by plate counting, between 104

and 106 CFU ml-1 (Jeanthon et al., 1988). They pointed out the beneficial effects in terms of

the percentage of larvae that metamorphosed to spat and spat size after 30 days; a possible

indirect beneficial effect was improved growth and development during the larval phase.

The group of Riquelme (Riquelme et al., 1996, Riquelme et al., 1997, Riquelme et al., 2001),

performed the most comprehensive assessment on the benefits of probiotic bacteria in

bivalve cultures. Their results demonstrated that a strain of Pseudoalteromonas haloplanktis

was effective against different bacterial species, including members of genus Vibrio. Further,

they found that strain 11 (Pseudomonas sp) (one strain among 506 isolates) was effective in

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the larval cultures of Argopecten purpuratus against pathogenic Vibrio anguillarum-related

(VAR) strain.

The term ‘probiotic’ was also used by Gibson et al. (1998) in a study on the protection of C.

gigas larvae by the strain A199 (Aeromonas media), against the infection by Vibrio tubiashii.

The strain A199 showed a broad spectrum of in vitro inhibition, including members of genus

Vibrio and Aeromonas. The conditioning of larvae with the probiotic strain (104 CFU ml-1)

allowed the survival of larval cultures inoculated with the pathogen (102,103 and 105 CFU ml-

1).

Nakamura et al. (1999) reported the beneficial effects of strain S21, isolated from seawater,

against pathogenic Vibrios, as demonstrated by the protection offered to Pacific oyster C.

gigas larvae against V. alginolyticus. These results demonstrated that the inoculation of

probiotic at 104 or 105 CFU ml-1 immediately after the pathogenic strain (105 CFU ml-1)

reduced the larval mortality from 91.6% to 53.1 and 78.0%, respectively. In any case, the

potential probiotic maintained their numbers in the medium, avoiding the risk of a

dangerous proliferation.

Kesarcodi-Watson et al. (2009) first screened probiotics for effective use with green shell

mussel larvae (Perna canaliculus), and has demonstrated the benefit of including test

animals in the initial screening stages. It should also be considered that when screening for

effective probiotics, it is best to use bacteria associated with or existing in the natural

environment of the host. Effective application in flow-through hatchery production

conditions is achieved by switching off the water flow for just 2 h with daily probiotic

addition. A later study of Kesarcodi-Watson et al. (2010) demonstrated that both strains of

Alteromonas macleodii 0444 and Neptunomonas sp. 0536, were two novel probiotics for

effective use on hatchery-reared Green shell mussel larvae, Perna canaliculus.

In general, the positive effect of probiotic bacteria in bivalve mollusk species was

demonstrated in those studies. However, there are some limitations to the use of probiotics

in aquaculture. Because, the most important limitation to the use of probiotics is that in

many cases they are not able to maintain themselves in sufficient numbers in the standing

microbial communities, and so they need to be added regularly and at high concentrations

making this technique less cost effective (Defoirdt et al., 2007b). Probiotics that were mostly

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selected in vitro based on the production of inhibitory compounds might fail to produce

these compounds in vivo. Also, food safety issues and difficulties regarding incorporation of

viable cells in feed, maintaining them stable during storage have put a constraint on the full-

scale application of probiotics (Merrifield et al., 2010).

2.6.5. Antivirulence therapy

Antivirulence therapy is a promising alternative approach to treating bacterial diseases.

Because bacteria use virulence factors to cause infection, preventing pathogens from

producing and using such factors is a promising strategy for disease control. This therapy is

thus based on a thorough understanding of the mechanisms that pathogenic bacteria use to

cause diseases (Defoirdt, 2014). The major advantage of antivirulence therapy is that there

will be less or no interference with non-target organisms, such as the commensal

microbiota. This is because antivirulence therapy specifically targets virulence gene

expression and regulation (Defoirdt, 2013, Defoirdt, 2014). Antivirulence therapy consists on

one hand of specifically inhibiting one or more virulence factors. On the another hand,

antivirulence therapy can interfere with the mechanisms that control the expression of

virulence factors. These mechanisms are for instance cell-to-cell communication (or quorum

sensing) and host-pathogen signaling. So for example, quorum sensing disrupting agents can

prevent bacteria from attaching to the host and are in this way effective in disease control

(Defoirdt, 2014).

2.7. The use of poly–β–Hydroxybutyrate (PHB) as a tool to control disease in aquaculture

seed production

2.7.1. Introduction

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates that accumulate

intracellularly as carbon and energy storage materials in numerous microorganisms, usually

when grown under limitations of oxygen, nitrogen, phosphate, sulphur, or magnesium and in

the presence of excess carbon (Anderson and Dawes, 1990, Valappil et al., 2007). PHA is

typically produced as a polymer of 103 to 104 monomers (Suriyamongkol et al., 2007) which

exists as discrete granules, with about 5 to 13 granules per cell and with diameters of 0.2 to

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0.5 μm (Figure 2.15) (Lee, 1996a, Sudesh et al., 2000). The properties of PHAs are similar to

those of conventional petrochemical-based synthetic thermoplastics and can hence

potentially replace them. Furthermore, they become completely degraded to carbon dioxide

and water under aerobic conditions, and to methane and carbon dioxide under anaerobic

conditions by microorganisms in the environment (Anderson and Dawes, 1990, Lee, 1996b).

Figure 2. 15: Pseudomonas putida cells accumulating PHB inclusion under electron

microscopy (Luengo et al., 2003).

PHAs can be classified as either short-chain-length (SCF) PHAs with C3-C5 hydroxyacids as

monomers or medium-chain-length (MCF) PHAs with C6-C16 hydroxyacids as monomers. The

SCF-PHAs have properties close to the conventional plastics (stiff and brittle) while MCF

PHAs are flexible, present low crystallinities, tensile strengths and melting points. Certain

bacteria can synthesize PHAs containing both SCF and MSF monomer units (Philip et al.,

2007). The molecular weight of PHAs varies between 2 × 105 and 3 × 106 Daltons (Figure

2.16). The composition and molecular weight of the synthesized polymer are governed by

two factors: the microbial strains and the provided substrate (mainly the carbon source)

(Valappil et al., 2007). The best-known member of the PHAs is poly-β-hydroxybutyrate

(PHB), containing repeat units of (R)-3 hydroxybutyrate (Lee, 1996b).

Figure 2. 16: General structural formula of poly–β–hydroxybutyrate

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2.7.2. The production of polyhydroxyalkanoates

Previous studies have shown that several bacteria can accumulate high levels of PHA per dry

cell weight (Kim et al., 1994, Wang and Lee, 1997). They can be divided into two groups, of

which the first includes Ralstonia eutropha, Protomonas extorquens, and Pseudomonas

oleovorans. These bacteria require limitations of essential nutrients (such as nitrogen,

magnesium, phosphorous or sulphur) while those of the second group, i.e., Alcaligenes latus

and Azotobacter vinelandii, require no such nutrient limitations (Lee, 1996b). Nonetheless,

the PHA content can be increased if a nutrient limitation is applied (Wang and Lee, 1997).

The production of PHAs can be performed from a wide variety of renewable resources,

including pure sugars (glucose, xylose, sucrose) (Quillaguamán et al., 2006, Quillaguamán et

al., 2007), biomacro-molecules (starch, cellulose) (Keenan et al., 2006, Halami, 2008), and

by-products (molasses, whey, wheat bran, corn steep liquor) (Ahn et al., 2000, Vijayendra et

al., 2007), as well as fossil resources (methane, mineral oil, lignite, hard coal) (Reddy et al.,

2003, Mothes et al., 2008), chemicals (acetate, propionic acid, butyric acid) (Shi et al., 1997)

and carbon dioxide (Ishizaki et al., 2001). The metabolic pathway of PHA synthesis in

bacteria from different carbon sources is summarized in Figure 2.17. The carbon source is

first transferred from the extracellular environment into the cells by a specific transport

system or diffusion. The carbon source is then converted into an (R)-hydroxyacyl-CoA

thioester (a substrate of the PHA synthase) by anabolic or catabolic reactions or both.

Finally, PHA synthases, which are the key enzyme of PHA synthesis, use (R)-hydroxyacyl-CoA

thioesters as the substrate and catalyzes the formation of PHA inclusions with a concomitant

release of coenzyme A (Steinbüchel and Valentin, 1995).

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Figure 2. 17: A schematic representation of the biosynthesis of PHA in bacteria (adapted from Steinbüchel and Valentin (1995))

Metabolic pathways and possible options for production of (R)-3-HB are described in Figure

2.18. For the production of (R)-3-HB, pathways leading to PHB production and degradation

have to be exploited. Recently, a metabolic pathway for the production of PHB and in vivo

hydrolysis to release (R)-3-HB in the culture supernatant was investigated (Shiraki et al.,

2006). Culture conditions would affect the metabolic shift of the PHB pathway; thereby

enhancing the extracellular production of (R)-3-HB in the culture supernatant. Furthermore,

it may be possible to introduce PHB knock-out in the PHB metabolic pathway of R. eutropha.

This process would involve the conversion of acetoacetyl-CoA to acetoacetate (in the

presence of acetoacetyl-CoA synthetase), which finally could be converted to (R)-3-HB.

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Figure 2. 18: Metabolic pathway for microbial production of (R)-3-hydroxybutyric acid (Tokiwa and Ugwu, 2007).

2.7.3. PHB production in Ralstonia eutropha

Ralstonia eutropha (formerly named Alcaligen eutrophus) has been studied most extensively

due to its ability to accumulate a large amount of PHB from pure carbon sources, for

example, glucose, fructose and acetic acid. Imperial Chemical Industries (ICI, UK) has been

producing PHB on a large scale from glucose, and poly3-hydroxybutyrate-co-3-

hydroxyvalerate (P(3HB-co-3HV)) from a mixture of glucose and propionic acid by the fed-

batch culture of R. eutropha. Initially, the cells are grown in a glucose-salts medium

containing calculated amounts of phosphate to support the desired amount of cell growth.

Cells encounter phosphate limitation after about 60 h and accumulate PHB during the next

40–60 h from supplied glucose. By controlling the glucose concentration at 10–20 g l-1 during

fed-batch culture the final cell mass, PHB concentration and PHB content of 164, 121 g l-1,

and 76%, respectively, is obtained for 50 h, resulting in a high productivity of 2.42 g (l-1 h-1)

(Kim and Chang, 1995). Several carbon sources other than glucose have also been used as

the substrate for PHB production by Ralstonia eutropha (Table 2.6).

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Table 2. 6: The evaluation of various PHB production systems reported for wild-type PHB

producing strains (Tokiwa and Ugwu, 2007).

Strain Carbon

substrate

PHB

analysis

Cultur

e time

(h)

Culture

mode

PHB

con.

(gl-1 )

PHB prod.

(gl-1h-1 )

PHB

conte

nt (%)

Reference

A. latus (DSM 1123) Sucrose GC 20 Feb-batch 98.7 4.94 88 Wang and Lee

(1997)

A. eutrophus

(NCIMB 11599)

Glucose GC 50 Feb-batch 121 2.42 76 Kim et al. (1994)

R.eutropha H16 Soybean

oil

GC 96 Feb-batch 95.8 1.00 76 Kahar et al.

(2004)

Azotobacter

chrococcum

Starch GC - Continuous 30.1 0.87 75 Du et al. (2001)

A. latus (DSM 1123) Sucrose GV 93 Feb-batch 4.9 0.05 63 Grothe et al.

(1999)

R. eutropha WSH3 Tapioca GC 59 Feb-batch 61.5 1.04 58 Kim and Chang

(1995)

A. latus (DSM 1123) Sucrose GC 18 Feb-batch 71.4 3.97 50 Yamane et al.

(1996)

CDW=cell dry weight; conc.= concentration; GC= gas chromatography; GV= gravimetric; prod.= productivity.

2.7.4. Mechanism of antibacterial activity of poly-β-hydroxybutyrate

Poly-β hydroxybutyrate (PHB) is a short chain fatty acid polymer of biological origin, which is

insoluble in water. PHB is degraded into water-soluble monomers or oligomers by the action

of PHB depolymerase enzymes secreted in the gastrointestinal passage of many aquatic

animals (Liu et al., 2010). PHB is produced by a wide variety of microorganisms as an

intracellular energy and carbon storage compound (Anderson and Dawes, 1990, Madison

and Huisman, 1999). PHB is deposited intracellularly in the form of inclusion bodies in the

fluid, amorphous state (Amor et al., 1991). After death and cell lysis, the polymer is released

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in a partially crystalline state (Doi et al., 1995). The ability to degrade extracellular PHB

depends on the secretion of extracellular PHB depolymerase enzymes and is widely

distributed in bacteria and fungi (Jendrossek, 1998, Jendrossek and Handrick, 2002). The

extracellular PHB depolymerase of Comamonas testosterone is well characterized; the

enzymes were found to hydrolyse PHB into β-hydroxybutyrate monomers (Mukai et al.,

1993, Kasuya et al., 1994).

PHB has been shown to effectively exhibit some antimicrobial, insecticidal and antiviral

activities (Tokiwa and Ugwu, 2007). Philip et al. (2007) mentions for example the commercial

product Nodax which is a copolymer containing mainly 3(HB) and small quantities of MCL

monomers. It can degrade anaerobically and hence can be applied as a coating for urea

fertilizers. It is used in rice fields as herbicide and insecticide. As such, it may also be applied

as a means to control infectious disease in aquaculture systems (Defoirdt et al., 2009). The

antimicrobial activity of β-hydroxybutyrate acid is thought to be comparable to that of other

short-chain fatty acids (SCFAs) (Defoirdt et al., 2007b). The main bacteriostatic and/or

bactericidal mechanism of SCFAs (and organic in general) is believed to be caused by the

undissociated form of the acid (Ricke, 2003). In the undissociated form, the short-chain fatty

acids can pass the cell membrane of bacteria and dissociate in the more alkaline cytoplasm,

thereby increasing the intracellular concentration of protons (Kashket, 1987, Cherrington et

al., 1991). Consequently, the cells have to spend energy in order to maintain the intracellular

pH at the optimal level. This energy cannot be used for other metabolic processes and

therefore, the growth of the cells is inhibited (Defoirdt et al., 2007b).

In fact, it has been suggested that short-chain fatty acids (SCFAs) could be useful as

biocontrol agents to control bacterial diseases in animal production and more specifically

aquaculture (Defoirdt et al., 2009). Several studies have demonstrated that SCFAs inhibit the

growth of enterobacteria like Salmonella Typhimurium, and Shigella flexneri (Cherrington et

al., 1991). In another report, the well-known bacterial storage compound PHB, a polymer of

the SCFA ß-hydroxybutyrate, was also shown to protect Artemia larvae from the virulent V.

campbellii strain. The degraded product can exert its beneficial effects like other SCFA do

(Defoirdt et al., 2007b). In several experiments with A. fransiscana, this approach increased

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the survival of the animals by up to 73% following a challenge with the pathogen V.

campbellii (Halet et al., 2007, Defoirdt et al., 2007b).

2.7.5. The immunostimulants properties of the Poly-β-hydroxybutyrate monomer

In addition to its antimicrobial function, SCFA butyrate also induces heat shock protein (Hsp)

25 in rat intestinal epithelial cells and protects the latter against oxidant injury (Ren et al.,

2001). HSPs are a group of highly conserved proteins of which expression is constitutive or

inducible under different conditions. Hsps, particularly Hsp70, have strong cytoprotective

effects and behave as molecular chaperones for maintaining proper protein folding,

disaggregating, and refolding misfolded protein as well as targeting damaged proteins for

degradation (Hishiya and Takayama, 2008, Tutar and Tutar, 2010). Besides these, Hsp70 also

generates protective immunity against many diseases as demonstrated in a wide variety of

animal models (Johnson and Fleshner, 2006). Baruah et al. (2015) suggested that PHB

conferred protection to Artemia host against V. campbellii by a mechanism of inducing heat

shock protein (Hsp) 70. Additionally, further findings also showed that this salutary effect of

PHB was associated with the generation of protective innate immune responses, especially

the pro-phenoloxidase and transglutaminase immune systems – phenomena possibly

mediated by PHB-induced Hsp70. From overall results, it can be concluded that PHB induces

Hsp70 production which might contribute in part to the protection of Artemia against

pathogenic V. campbellii (Baruah et al., 2015).

Yik Sung et al. (2007) and Baruah et al. (2013) demonstrated a correction between the

amount of induced Hsp70 and the degree of protective immune responses against diseases

in animals. Baruah et al. (2015) determined that PHB not only regulates the expression of

innate immune related genes in Artemia but also increases the activity of phenoloxidase in

Artemia treated with PHB and simultaneously challenged with V. campbellii. Further, Suguna

et al. (2014) recently reported the immunostimulatory efficacy of poly-β hydroxybutyrate–

hydroxyvalerate (PHB–HV) extracted from Bacillus thuringiensis B.t.A102 in another

aquaculture species, the tilapia Oreochromis mossambicu. These results revealed that all the

doses of PHB–HV supplementation in feed were effective in stimulating nonspecific immune

mechanisms.

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As a conclusion from the research mentioned above, it can be stated that SCFAs and β-

hydroxybutyrate acid showed good potential as antimicrobial and immunostimulant

compounds to fight infections in aquaculture. However, the mode of action of PHB in

aquaculture species needs to be further studied e.g. in relation to its capacity to regulate the

immune response.

2.7.6. The use of poly-β-hydroxybutyrate in aquaculture

Bacterial disease outbreaks are considered a significant constraint to the development of the

aquaculture sector, as well terrestrial animal production. The ban on the use of antibiotics to

control diseases in these production sectors has challenged researchers throughout the

world to look for alternative biocontrol strategies (Nicolas et al., 2007, Sapkota et al., 2008).

As PHB has been shown to effectively exhibit some antimicrobial, insecticidal and antiviral

activities (Tokiwa and Ugwu, 2007) it is increasingly being tested as an antimicrobial

compound to control bacterial infection in aquaculture research, especially in larval rearing.

Recently, a high number of trials with PHB aquatic animals have been done, demonstrating

positive effects on survival, growth rate and disease resistance. It also seems to act as an

antimicrobial agent and specially enhances “positive” shifts in the microbial community

composition in the digestive tract of aquaculture animals.

Defoirdt et al. (2007b) started to investigate whether poly-β-hydroxybutyrate (PHB) could be

used as an elegant method to deliver SCFAs to the brine shrimp gut. The addition of 1000 mg

L-1 commercial PHB particles (average diameter 30 μm) to the culture water offered a

complete protection (no significant mortality when compared with uninfected nauplii) from

the pathogenic V campbellii (Defoirdt et al., 2007b). In a follow-up study, it was shown that

the addition of 107 CFU ml-1 of PHB-containing Brachymonas bacteria (corresponding to

∼10 mg L-1 PHB) also completely protected the shrimp from the Vibrios (Halet et al., 2007).

Based on these results it was hypothesized that the PHB polymer is (at least partially)

degraded to β-hydroxybutyrate in the Artemia gut and that the release of this SCFA protects

the shrimp from the pathogen (Defoirdt et al., 2007a). Recently, a number of studies have

found a positive effect of PHB treatment on the survival of either the larval or juvenile stage

of a number of aquaculture species. For example, Nhan et al. (2010) reported the increased

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Chapter 2 Literature review

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survival and growth in the larvae of the giant fresh water prawn, following PHB enrichment

at 5 g L-1 in combination with/or highly unsaturated fatty acids (HUFA). Similar beneficial

effects have been reported in another study (Ludevese-Pascual personal communication,

Ugent) in which survival rates of tiger shrimp Penaeus monodon postlarvae increased via

PHB incorporated in Artemia nauplii. Laranja et al. (2014) reported that PHB is accumulated

in Bacillus spp, improving the survival, growth and robustness of Penaeus monodon

(Fabricius, 1798) postlarvae. In other crustacean aquaculture species, enhanced growth,

survival and protection from Vibrio anguillarum was found in the zoea larvae of the Chinese

mitten crab (Eriocheir sinensis) (Sui et al., 2012). Beneficial effects of PHB have also been

reported in fish culture systems. For example, (Situmorang et al., 2016) reported that PHB

also acted as a protecting agent for Nile tilapia larvae, resulting in a 20% increase in larval

survival following a gnotobiotic challenge test with the pathogen Edwardsiella ictaluri

gly09R.

Further investigations are required to verify the effect of PHB in the different animal species,

with focus on using PHB supplementations in fish diets. De Schryver et al. (2010) replaced

the sea bass (Dicentrachus labrax) diet partially with 2%, 5%, and 10% of the dry feed weight

with PHB. They documented that PHB could serve as an energy source in sea bass juveniles

(as in a diet with 100% PHB, survival was considerably higher than with starved animals) and

significantly increased growth rate and decreased FCR when present at 2% and 5% in the

diets. In addition, a reduction of the gut pH from 7.7 to 7.2 was observed, suggesting that

PHB can be degraded in the intestine, leading to an increased production of short-chain fatty

acids. Based on molecular analysis, higher dietary PHB levels induced larger changes in the

bacteria community composition and the highest bacteria range-weighted richness in the

fish intestine was observed in PHB treatments.

In the later experiment, Najdegerami et al. (2012) confirmed the positive effect of PHB on

growth performance of Siberian sturgeon (Acipenser baerii) fingerlings and microbial

community in its gastrointestinal tract. The results indicated that a 2% replacement in the

diet with PHB improved weight gain, specific growth rate (SGR) and survival in the sturgeon

fingerlings during the 10-week experimental period. Juveniles fed with 2% PHB enriched diet

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exhibited the highest intestinal microbial species richness. The development of bacteria

belonging to the genera Bacillus and Ruminococcaceae was promoted.

Based on current results, the use of PHB looks promising for aquaculture, especially in

improving growth and survival in larval culture systems. However, in terms of effectiveness,

the use of bacteria cells that contain PHB-A (amorphous PHB particles) can be considered as

an effective alternative for PHB-C (crystalline) particles (Halet et al., 2007). It is reported that

the extraction of PHB from the cells amount to about 30% of the total production cost of the

pure/ crystalline PHB-C (Mudliar et al., 2008). Consequently, the absence for the need of

PHB extraction in the application of amorphous PHB will considerably decrease the

application costs of PHB. It was estimated that the price of PHB contained within bacterial

cells will be about 40% lower than that of pure extracted crystalline PHB (De Schryver et al.,

2010). Overall, more research is needed on PHB degradation, metabolism and immune

response, in different aquaculture species in order to effectively and efficiently apply PHB as

immunostimulants/additives in aquaculture production.

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Application of poly-β-hydroxybutyrate (PHB) in

mussel larviculture

Nguyen Van Hung1,2, Peter De Schryver1, Tran Thanh Tam1, Linsey Garcia-Gonzalez3, Peter Bossier1 and Nancy Nevejan1

1Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Rozier 44, B-9000 Gent, Belgium

2 Research Institute for Aquaculture no.3, 33 Dangtat, Nhatrang, Vietnam

3 Flemish Institute for Technological Research (VITO), Boeretang 200, 2400 Mol, Belgium

Published in Journal of Aquaculture (2015) 446, 318 - 324

CHAPTER 3: APPLICATION OF POLY-Β-HYDROXYBUTYRATE (PHB) IN MUSSEL LARVICULTURE

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Chapter 3 Application of PHB in mussel larviculture

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Abstract

In this study, the effect of poly-β-hydroxybutyrate (PHB) delivery on the performance

(survival, growth and metamorphosis) of blue mussel (Mytilus edulis) larvae was studied for

the first time. Upon addition of PHB in either crystalline (i.e. extracted from the bacterial

cell) or amorphous (i.e. still contained in the bacterial cell) form to a standard algal diet at

concentrations of 0.1 mg L-1, 1.0 mg L-1 and 10.0 mg L-1, no significant improvement in

growth performance or metamorphosis was observed. However, a concentration of 1.0 mg

L-1 amorphous PHB almost doubled the survival of the larvae in three independent

experiments. A total of 22 PHB-degrading bacterial strains could be isolated from the PHB

treated mussel larvae of which 16 could be characterized to belong to the genus

Pseudoalteromonas. The application of these in combination with PHB however, did not

further increase the culture performance of the mussel larvae. Where previous studies

suggested that the PHB effect could be related to a change in the intestinal microbial

community of the treated animals, molecular fingerprinting analysis of the mussel larvae

associated microbiota did not show a relationship between changes in the microbiota

composition and the improved survival following PHB treatment. Overall, this study showed

for the first time that PHB can be applied in blue mussel larviculture to increase the survival

of the cultured animals.

Keywords: Mussel larvae, crystalline PHB, amorphous PHB, PHB-degrading bacteria, Mytilus

edulis

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Chapter 3 Application of PHB in mussel larviculture

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3. 1. Introduction

Mass mortalities of bivalve larvae caused by bacterial infections are reported regularly

(Nicolas et al., 1996, Romalde et al., 2013). For example, mass mortality of early stage oyster

larvae has been attributed to Vibrio tubiashii infections in 2006 and 2007, resulting in 59%

loss in hatchery production (Elston et al., 2008). To eliminate pathogenic bacteria, many

bivalve hatcheries use different water treatments such as filtration, pasteurization,

ozonation and UV radiation (Verschuere et al., 2000). The use of chemotherapeutic agents is

widespread in mollusk hatcheries, although showing inconsistent results (Prado et al., 2010).

In addition, many hatcheries use antibiotics to culture the larval phases (Nicolas et al., 1996,

Sugumar, 1998, Uriarte et al., 2001, Cabello, 2006). However, due to rising concerns about

public health and environmental safety issues, the use of antibiotics are being discouraged

worldwide (Schneider, 2003). Attempts have been made to enhance the culture of larval

bivalves by controlling the microbiota in the culture water to mitigate the effects of

pathogens in bivalve hatcheries. Of these methods, prebiotics and probiotics have gained a

lot of interest recently (Gibson et al., 1998, Ruiz-Ponte et al., 1999, Riquelme et al., 2000,

Kesarcodi-Watson et al., 2008, Kesarcodi-Watson et al., 2010, Prado et al., 2010). For

example, the addition of the probiotic Alteromonas sp. (CA2 strain) resulted in an enhanced

survival and growth rate of Pacific oyster larvae (Douillet and Langdon, 1993) while Riquelme

and colleagues (Riquelme et al., 1997, Riquelme et al., 2000, Riquelme et al., 2001) used

three strains of probiotic bacteria - Vibrio sp. C33, Pseudomonas sp. 11 and Bacillus sp. B2 –

to enhance the survival of scallop (Argopecten purpuratus) larvae. As illustrated by these

examples, the search for novel sustainable alternatives for maintaining healthy bivalve

larvae has become increasingly important.

The bacterial storage compound poly-β-hydroxybutyrate (PHB) has been applied as a

prebiotic and proved to act beneficially for a variety of aquatic animals (De Schryver et al.,

2010). PHB is a natural polymer that is accumulated by a wide variety of microorganisms

that produce this polymer of the fatty acid β-hydroxybutyrate as an intracellular energy and

carbon storage compound (Anderson and Dawes, 1990). Within a bacterial cell, PHB is

present in a non-crystalline amorphous state whereas upon lysis of the cell the PHB becomes

(partially) crystalline (Jendrossek and Handrick, 2002). The use of PHB for aquaculture

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Chapter 3 Application of PHB in mussel larviculture

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purposes was introduced in 2007 when Defoirdt et al. (2007b) and Halet et al. (2007)

described the effectiveness of the compound to protect Artemia franciscana larvae against

Vibrio infection. It was suggested that this was due to the gastrointestinal production of the

monomer β-hydroxybutyrate (β-HB) that had an anti-pathogenic activity. Since then, the

application of PHB has been successfully tested on a variety of aquaculture species: PHB

significantly increased the growth rate of larvae of giant freshwater prawn (Macrobrachium

rosenbergii) when supplied through live food (Nhan et al., 2010) and of juvenile European

seabass (Dicentrarchus labrax) when included in the diet at 2 and 5% (w/w) (De Schryver et

al., 2010). Survival and growth rate of Siberian sturgeon (Acipenser baerii) fingerlings was

improved when included in the diet at 2% (w/w) (Najdegerami et al., 2012). It also protected

zoea larvae of the Chinese mitten crab (Eriocheir sinensis) against V. anguillarum (Sui et al.,

2012) and larvae of giant freshwater prawn against V. harveyi BB120 infection (Thai et al.,

2014).

The ability to produce extracellular PHB depolymerase enzymes is widely distributed among

bacteria and fungi (Jendrossek and Handrick, 2002). Liu et al. (2010) isolated PHB-degrading

bacteria from the gastrointestinal tract of juvenile European sea bass, juvenile Siberian

sturgeon and giant freshwater prawn larvae. They evaluated their efficiency to increase the

beneficial effect of PHB in a synergetic approach and the results provided perspectives to

improve the gastrointestinal health of aquatic animals.

In this study, the impact of PHB on the culture performance of blue mussel larvae was

investigated for the first time. PHB was supplied in crystalline and amorphous form at

different concentrations to the culture water of blue mussel larvae (Mytilus edulis) and the

survival, growth and metamorphosis success of the larvae was assessed. PHB-degrading

bacteria associated with the larvae were isolated and supplemented, and changes in the

intestinal microbial community composition of the larvae were investigated. The table below

gives an overview of the experiments. Experiment 1 lasted for 18 days (22 March – 9 April,

2013); experiment 2 lasted for 14 days (8 May – 22 May, 2013) and experiment 3 for 18 day

(22 June – 10 July, 2013).

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Chapter 3 Application of PHB in mussel larviculture

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Table 3. 1 Overview of the experiments

Experiment (Exp.)

Treatment

Periods of experiment Evaluation

Larvae spat

Exp. 1

(n=3)

Effect of different

PHB form

Control

PHB-C 0.1 mg L-1

PHB-C 1.0 mg L-1

PHB-A 0.1 mg L-1

PHB-A 1.0 mg L-1

18 day

Sample for

MC analysis

15 day

- Larvae performance

- Spat performance

- Microbial community

(MC)

Exp. 2

(n=5)

Optimization of

PHB-A

concentration in

larvae culture

Control

PHB-A 1.0 mg L-1

PHB-A 10.0 mg L-1 14 day

Sample for

MC analysis

15 day

- Larvae performance

- Spat performance

- MC

Exp.3

(n=4)

Impact of PHB

degrader bacteria

in larvae culture

Control

PHB-A 1.0 mg L-1

PHB-A 1.0 mg L-1

+

PHB degrader

PHB degrader

18 day 15 day

- Larvae performance

- Spat performance

3. 2. Materials and methods

3. 2.1. Crystalline PHB and amorphous PHB

Two forms of PHB were used in this study. Crystalline PHB particles (PHB-C) were obtained

from Goodfellow (Huntingdon, England) and amorphous PHB (PHB-A) consisting of a freeze-

dried Ralstonia eutropha culture containing 75% cell dry weight of PHB was obtained from

VITO (Mol, Belgium). Both of them were ground and sieved through a 30 µm mesh before

addition into the culture water to make sure that the particles could be filtered out of the

water by the larvae.

3. 2. 2. Detection of ingested PHB by fluorescence microscopy

Before performing the experiments, fluorescence microscopy was used to verify the ability

of mussel larvae to ingest PHB. For that purpose crystalline PHB was stained with Nile blue.

500 µl Nile blue A (NBA) was added to 500 mg sonicated PHB in 30 ml of deionized water for

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Chapter 3 Application of PHB in mussel larviculture

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20 minutes. It was consequently centrifuged at 5000 rpm for 10 minutes. The solution was

resuspended to wash 3 times with deionized water. Four day post-hatching D-larvae were

starved for 24 h to eliminate all algae present in the intestines. Then, the larvae were

supplied with 30 µm sieved and Nile Blue A stained PHB (Ostle and Holt, 1982) at a

concentration of 1 g L-1 in 0.2 µm filtered natural seawater (FNSW). After two hours, the

larvae were collected on a 60 µm sieve, rinsed with seawater to remove all PHB particles

from the outer surface of the larvae and examined under a fluorescence microscope

(Axioskop II, Zeiss, Jena, Germany) operating at 488 nm wavelength and using a total

magnification of 630 X. The microscope was equipped with a Peltier-cooled single-chip

digital camera (Orca Illm; Hamamatsu, Massay, France) connected to a PC. Larvae that were

not supplied PHB served as control.

3. 2. 3. Experimental design

3. 2.3.1. Mussel larvae source and experimental conditions

Broodstock animals were supplied by the commercial mussel producer Roem van Yerseke in

The Netherlands. Upon arrival in the laboratory, they were rinsed with FNSW. Spawning was

induced by thermal shocks, by emerging the animals alternately in water baths of 15°C and

25 °C every 15 minutes. Individual matured males and females were identified at the time of

gamete release and isolated into individual beakers. Eggs and sperm of individual females

and males, respectively, were pooled before fertilization was carried out. Fertilized eggs

were regularly stirred with a plunger during 30 min before being rinsed with FNSW and put

in a 20 L incubator rectangular tank. Two days after fertilization, hatched D-larvae were

concentrated on a sieve (60 µm), rinsed with FNSW and counted before stocking in 8L Züger

bottles (Urbányi et al., 2008). The larvae from all treatments were fed every 2 days with an

algae diet consisting of Isochrysis galbana and Chaetoceros calcitrans (1:1, based on cell

numbers) for the first 4 days. From day 4 onwards Tetraselmis suecica was added as well

(1:1:1, based on cell numbers). The total concentrations gradually increased from 3 x 104 CFU

mL-1 at the start of the experiment to 1 x 105 CFU mL-1 at the end of the experiment in steps

of 1 x 104 CFU mL-1 per 2 days.

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Water was changed completely every two days. At that occasion, feed was administered and

samples were taken for larval survival and height measurements. During the experiments,

gentle bottom aeration was provided to supply oxygen and enhance larvae suspension. The

water temperature of the larvae culture was on average 17 ± 0.5 °C during experiment 1 and

3 and 19 ± 0.5 °C during experiment 2.

Three different experiments were performed:

- In experiment 1, the effects of PHB-C and PHB-A were compared. The larvae were stocked

in the Züger bottles at a density of 5 larvae mL-1. The following 5 treatments were conducted

in triplicate for 18 days: (1) Control, consisting of the algae diet only, (2) PHB-C 0.1,

consisting of adding crystalline PHB at 0.1 mg L-1 to the algae diet, (3) PHB-C 1.0, consisting of

adding crystalline PHB at 1.0 mg L-1 to the algae diet, (4) PHB-A 0.1, consisting of adding

amorphous PHB at 0.1 mg L-1 to the algae diet, and (5) PHB-A 1.0, consisting of adding

amorphous PHB at 1.0 mg L-1 to the algae diet. The parameters as described under 3.2.3.2

were assessed on Day 14 and on Day 18 post-hatching. At the end of experiment 1, PHB-

degrading bacteria were isolated from the mussel larvae (see 3.2.4).

- In experiment 2, the optimal concentration of PHB-A for the mussel larvae was determined

as PHB-A showed better effects than PHB-C in experiment 1. The larvae were stocked at a

density of 10 larvae mL-1 in the Züger bottles and the following 3 treatments were conducted

in quintuplicate for 14 days: (1) Control, consisting of the algae diet only, (2) PHB-A 1.0,

consisting of adding amorphous PHB at 1.0 mg L-1 to the algae diet and (3) PHB-A 10 mg L-1,

consisting of adding amorphous PHB at 10 mg L-1 to the algae diet. The parameters as

described under 3. 2.3.2 were assessed on Day 10 and on Day 14 post-hatching.

- In experiment 3, the optimal dose of PHB-A from experiment 2 was combined with the

strongest bacterial PHB degrader (isolate ARC4B5 obtained from experiment 1, see 3.2.4) to

examine whether the effects of amorphous PHB could be further enhanced. Larvae were

stocked at a density of 10 larvae mL-1 in the Züger bottles and the following 4 treatments

were conducted in quadruplicate for 18 days: (1) Control, consisting of the algae diet only,

(2) PHB-A 1.0, consisting of adding amorphous PHB at 1.0 mg L-1 to the algae diet, (3) PHB-A

1.0 + PHB degrader, consisting of adding amorphous PHB at 1.0 mg L-1 and 106 colony

forming units (CFU) mL-1 of ARC4B5 to the algae diet and (4) PHB-degrader, consisting of

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Chapter 3 Application of PHB in mussel larviculture

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adding 106 CFU mL-1 of ARC4B5 to the algae diet. The parameters as described under 3.2.3.2

were assessed on Day 14 and on Day 18 post-hatching.

3. 2.3.2. Larval survival, height measurement, and settlement

The survival and growth of the larvae in experiments were determined as follows. For

sampling, all larvae were collected on a 60 to 90 µm sieve depending on the average larval

size, and concentrated in 1.5 L of FNSW. A plunger was used to distribute larvae

homogenously in the beaker before taking a subsample of 2 mL in four replicates. Dead and

living larvae were distinguished under a light microscope (Euromex, Holland) using Lugol’s

solution (Sullivan and Gifford, 2009). Deterioration of organ structure and empty shells

denoted moribund or dead individuals. The growth was determined by measuring the shell

height of fifty larvae that were randomly sampled from a 2 mL subsample. The shell height,

being the largest distance from the ventral side to the straight-hinge, was measured using

the automated image analysis software Clemex Vision PE version 3.6.002 based on images

obtained with a light microscope at a magnification of 400x, equipped with a MicroPublisher

3.3 camera (QImaging, China) directly connected to the computer with the software.

At the end of each experiment, all larvae from each Züger bottle were harvested on a 150

µm sieve. After re-suspension, 1000 larvae from each replicate were stocked in a

downweller, consisting of a PVC cylinder (Ø 12cm, H 14cm) provided with a bottom sieve of

150 µm. The downwellers were placed in a rectangular tank (water bath) containing 60 L of

FNSW of 17 ± 0.5 °C, making sure that each tank contained 1 replicate of each treatment.

The larvae were fed a combination of the three microalgae species that were administered

during the larval stage at a concentration of 1 x 105 CFU mL-1 without any addition of PHB

particle or amorphous PHB. The water was changed completely every 2 days and after 15

days, settlers (young spat) were counted and the shell height of fifty settlers from each

replicate was measured.

3. 2.4. Isolation, culture and identification of PHB-degrading strains

At the end of experiment 1, larvae from each PHB treatment were collected to isolate PHB-

degrading bacteria from the intestinal tract. About ten thousand larvae from each replicate

were collected on a sterile 100 µm sieve and rinsed twice with 0.2 µm filtered autoclaved

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Chapter 3 Application of PHB in mussel larviculture

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artificial sea water (FAASW) (35 g L-1 Instant Ocean®, Aquarium Systems Inc., Sarrebourg,

France). The larvae were transferred to a sterile plastic bag containing 2 ml of FAASW and

homogenized with a stomacher blender (400SN, Seward Medical, London, UK) for 5 min. A

70 µL aliquot of the homogenate was added either to 10 mL marine broth (MB - DifcoTM

Marine Broth 2216) to boost bacterial growth prior to PHB degrader selection or to 10 mL

PHB minimal medium (FAASW containing 0.2 g L-1 NH4Cl and 1.0 g L-1 crystalline PHB

particles, average diameter 30 µm) to directly select for PHB degraders. The cultures were

incubated at 28°C on an orbital shaker (130 rpm) for 1 and 3 days, respectively. Both cultures

were sub-cultured twice by transferring 1 ml of culture medium into 100 mL fresh PHB

minimal medium (which was incubated at 28°C on an orbital shaker (130 rpm) for 3 days)

before being plated on marine agar where single colonies could be picked. Morphologically

different colonies were incubated in PHB minimal medium for 72h and subsequently purified

by streak plating on marine agar resulting in 22 isolates. Extracellular PHB depolymerase

production was assessed qualitatively by spotting the isolates on PHB agar (being PHB

minimal medium supplemented with 15 g L-1 agar) or PHB agar supplemented with 10% LB

medium as the carbon sources (Defoirdt et al., 2007b, Liu et al., 2010). The plates were

incubated at 28°C and examined daily for the presence of clearing zones around the

colonies. PHB-degrading bacteria were stored in 20% glycerol at -80 °C until further use.

A 1465 base pair fragment of the 16S rRNA gene of the 22 isolated PHB-degrading strains

was amplified using the universal primers 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R

(5’-CGGTTACCTTGTTACGACTT-3’) (Jiang et al., 2006). PCR was performed using a GeneAmps

PCR system 2700 thermal cycler using the following program: 95°C for 5 minutes, 30 cycles

of 95°C for 1 minute, 50°C for 1 minute, 72°C for 2 minutes and finally an extension period of

72 °C for 10 minutes. The resulting PCR product was purified using a Wizard® SV Gel and PCR

Clean-Up System (Promega, The Netherlands) and sequenced at the Center for Medical

Genetics (Ghent University Hospital, Belgium) using the PCR primers. The nucleotide

sequences were analyzed using the VECTOR NTI ADVANCE program, version 11.5 and

homology searches were completed with the BLAST server of the National Centre for

Biotechnology Information using the BLAST algorithm (Altschul et al., 1997) for comparison

of the nucleotide query sequence against a nucleotide sequence database (BLASTN).

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3. 2.5. Molecular fingerprinting of larval associated bacterial community

3. 2.5.1. DNA extraction and PCR amplification

From each replicate, about five thousand mussel larvae were collected on a 100 μm sieve at

the end of the experiments 1 and 2 and rinsed three times with FAASW. The larvae were

transferred to a sterile 2 ml tube and stored at -20°C until further processing. The extraction

of DNA from the samples was carried out with a DNeasy Blood and Tissue Kit (Qiagen, The

Netherlands) according to the manufacturer’s instruction. PCR amplification targeting the V3

region of the 16S rRNA gene was conducted as described by Boon et al. (2002) using

bacterial primer 338f (5′-ACTCCTACGGGAGGCAGCAG-3′) with a 40-base GC clamp attached

to its 5′ end and universal primer 518r (5′-ATTACCGCGGCTGCTGG-3′).

3. 2.5.2. Denaturing gradient gel electrophoresis (DGGE)

A Bio-Rad D Gene system (Bio-Rad, Hercules, CA, USA) was used to perform DGGE analysis

(Boon et al., 2002). The denaturing gradient of the gel ranged between 45% and 60%.

Electrophoresis was performed with a constant voltage of 38 V at 60°C for 16 h. Bands were

visualized using a UV transilluminator after staining the gel for 20 minutes in a 1% gel-red

solution.

3. 2.5.3. Processing DGGE images and statistical analysis

The DGGE patterns were processed with BIONUMERICS software version 5.0 (Applied Maths,

Sint-Martens-Latem, Belgium) and analyzed as described by Bakke et al. (2013). A 1% band

position tolerance limit was maintained for considering bands as being identical. As DGGE

analysis of the DNA of the algae used as feed also resulted in bands, the peak areas in each

sample pattern located at the position of the algal bands were deleted prior to analysis of

the gels.

3. 2.6. Statistics

The survival data of larvae and spat were arcsine transformed to satisfy the homogeneity of

variance requirement. The survival, height and the settlement success of the mussel larvae

in the different treatments were compared by one-way ANOVA analysis using SPSS (version

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Chapter 3 Application of PHB in mussel larviculture

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16.0). A Duncan post-hoc test was used for identification of significant differences between

the means (p ≤ 0.05). For analysis of the DGGE patterns from experiment 1 and 2, a matrix of

Bray – Curtis similarities (Bray and Curtis, 1957) between the samples from each experiment

(all loaded on a single DGGE gel) was constructed using the software Primer6 (Bakke et al.,

2013). A non-metric multidimensional scaling (NMS) plot was constructed using the software

SPSS (version 20.0). To identify significant differences between the microbial communities in

the different treatment, an ANOSIM analysis of the Bray-Curtis similarity values was

performed (Clarke, 1993) using Primer6. The ranked similarity R was determined by

performing 999 permutations.

3. 3. Results

3. 3.1. PHB ingestion by mussel larvae

Epifluorescence images of larvae showed that larvae fed with Nile Blue A stained PHB-

particles were brightly fluorescent in the stomach area (Figure 1). Fluorescence was already

visible 1 hour after supplementation of PHB but maximum intensity was reached after 2

hours. Fluorescence of the stained PHB lasted for 5 hours.

Figure 3. 1: Light (A and B) and epifluorescence microscopy (C and D) images of starved larvae (A and C) and larvae fed with Nile Blue A stained PHB for 2 hours (B and D)

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3. 3.2. Larval survival and growth

In experiment 1, the larvae fed the PHB-A 1.0 mg L-1 diet survived significantly better than

the larvae from the control treatment at day 14 and day 18 (Table 3. 2). The larvae from the

PHB-C 0.1, PHB-C 1.0 and the PHB-A 0.1 treatment tended to survive better than the larvae

from the control treatment, but the differences were not significant anymore on day 18. At

the onset of the experiment, the larvae had an average shell height of 70 µm. At 14 days and

18 days post hatching, the larvae supplied with PHB in either crystalline or amorphous form

were not bigger than the control larvae.

There were no significant positive effects of PHB on the settlement success of the mussel

spat (Table 3. 3). There was also no significant difference in spat size between the

treatments, although the spat of the control treatment had a tendency to be larger than the

spat from the PHB treatments.

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Table 3. 2: Survival (%) and shell height (µm) of mussel larvae subjected to different PHB treatments in experiment 1 and 3 (Day 14 post-hatching and Day 18 post-hatching) and in experiment 2 (Day 10 post-hatching and Day 14 post-hatching)

Treatment Survival Shell height Survival Shell height

Experiment 1 (n = 3) Day 14 post-hatching Day 18 post-hatching

Control 33.9 ± 6.1a 157.5 ± 3.1a 25.9 ± 8.9 a 185.5 ± 1.9a

PHB-C 0.1 mg L-1 56.2 ± 5.6b 146.2 ± 0.8a 35.4 ± 6.4ab 187.3 ± 0.2a

PHB-C 1.0 mg L-1 50.4 ± 4.1ab 148.2 ± 7.0a 32.3 ± 2.2ab 183.5 ± 8.4a

PHB-A 0.1 mg L-1 56.0 ± 4.0 b 145.1 ± 2.8a 39.7 ± 7.1ab 197.4 ± 1.3a

PHB-A 1.0 mg L-1 58.3 ± 6.9b 142.6 ± 2.4a 50.2 ± 6.2b 180.8 ± 4.5a

Experiment 2 (n = 5) Day 10 post-hatching Day 14 post-hatching

Control 42.9 ± 3.0a 160.5± 2.3b 26.5 ± 1.9a 177.1 ± 1.0b

PHB-A 1.0 mg L-1 53.8 ±1.2b 159.4 ± 2.0b 40.3 ± 2.4b 177.9 ± 3.4b

PHB-A 10.0 mg L-1 39.4 ± 2.5a 99.3 ± 0.9a 23.0 ± 0.8a 113.9 ± 5.7a

Experiment 3 (n = 4) Day 14 post-hatching Day 18 post-hatching

Control 49.0 ± 2.5a 138.7 ± 1.0a 33.5 ± 3.0a 189.8 ± 4.4a

PHB-A 1.0 mg L-1 56.5 ± 5.2ab 159.8 ± 4.9b 47.7 ± 4.0b 186.8 ± 3.5a

PHB-A 1.0 mg L-1 +

PHB degrader 59.3 ± 4.5ab 122.2 ± 3.0c 31.6 ± 6.0a 149.1 ± 0.5b

PHB degrader 68.8 ± 4.8b 122.8 ± 3.0c 32.9 ±3 5.5a 141.5 ± 3.0b

Values represent means ± standard error. Data in the same column and within an experiment that are labelled with a different superscript letter are significantly different (p≤0.05)

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Table 3. 3: Settlement success (%) and shell height (µm) of mussel spat subjected to different PHB treatments in experiment 1, 2 and 3

Treatment Settlement success Shell height

(µm)

Experiment 1 (n = 5)

Control 41.4 ± 10.8a 388.2 ± 5.3a

PHB-C 0.1 mg L-1 43.3 ± 2.7a 347.2 ± 18.1a

PHB-C 1.0 mg L-1 27.7 ± 4.2a 352.9 ± 15.7a

PHB-A 0.1 mg L-1 43.0 ± 9.6a 365.5 ± 3.4a

PHB-A 1.0 mg L-1 51.2 ± 5.1a 352.1 ± 13.8a

Experiment 2 (n=5)

Control 35.5 ± 2.1a 433.8 ± 11.3a

PHB-A 1.0 mg L-1 30.5 ± 4.9a 428.9 ± 9.0a

PHB-A 10.0 mg L-1 24.1 ± 3.4a 418.9 ± 14.6a

Experiment 3 (n = 4)

Control 20.4 ± 7.3ab 375.6 ± 13.2a

PHB-A 1.0 mg L-1 34.3 ± 4.0b 333.6 ± 5.2ab

PHB-A 1.0 mg L-1 + PHB degrader 15.9 ± 3.9a 314.0 ± 4.5bc

PHB degrader 13.4 ± 1.4a 279.3 ± 16.9c

Values represent means ± standard error. Data in the same column and within an

experiment that are labelled with a different superscript letter are significantly different

(p≤0.05)

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Chapter 3 Application of PHB in mussel larviculture

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Figure 3. 2: Size class distribution of mussel larvae on day 14, subjected to different feeding regimes in experiment 2 (n =5)

Similar to experiment 1, the PHB-A 1.0 mg L-1 treatment in experiment 2 showed a

significantly higher survival as compared to the control treatment while the growth was not

significantly different (Table 3.2). The addition of 10 mg L-1 PHB-A to the rearing water

induced no difference in survival of the mussel larvae as compared to the control, but

significantly suppressed the growth. A total of 92% of the larvae in this treatment were

smaller than 180 µm while this was only 45% and 46% in the control treatment and the PHB-

A 1.0 mg L-1 treatment, respectively (Figure 3. 2). There were no significant effects of PHB-A

on the settlement success and the growth of the mussel spat (Table 3. 3).

The addition of the PHB-degrading isolate ARC4B5 in experiment 3 did not enhance the

positive effect of PHA-A 1.0 mg L-1 on the larval survival (Table 3. 2). On the contrary, the

presence of the PHB-degrader significantly decreased the growth of the larvae and the

settlement success. The resulting spat were also smaller (Table 3. 3).

Based on the results of Table 3.2 and Table 3.3, the total yield of mussel spat under

difference experimental conditions was calculated Table 3.4

0

20

40

60

80

100

Per

cen

tage

(%

)

Treatment

>=220

>=200- 220

>=180 - 200

<180

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Chapter 3 Application of PHB in mussel larviculture

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Table 3. 4. The final yield of mussel spat under different experimental conditions

Treatment Yield

(from egg to spat, %)

Experiment 1 (n = 5)

Control 10.7

PHB-C 0.1 mg L-1 15.3

PHB-C 1.0 mg L-1 8.9

PHB-A 0.1 mg L-1 17.0

PHB-A 1.0 mg L-1 25.7

Experiment 2 (n=5)

Control 9.4

PHB-A 1.0 mg L-1 12.3

PHB-A 10.0 mg L-1 5.5

Experiment 3 (n = 4)

Control 6.8

PHB-A 1.0 mg L-1 16.3

PHB-A 1.0 mg L-1 + PHB degrader 5.0

PHB degrader 4.4

3. 3.3. Isolation and identification of PHB-degrading bacteria

A total of 16 different isolates were obtained from the mussel larvae fed with either

crystalline PHB or amorphous PHB. A homology search revealed that all isolates belonged to

the genus Pseudoalteromonas. Isolate ARC4B5 was most closely related to the marine

bacterium Pseudoalteromonas species Strain X153 (GenBank accession number AJ581533)

and showed the highest PHB-degrading capacity according to a PHB depolymerase agar

assay (data not shown). Therefore it was selected for use in experiment 3.

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The nucleotide sequences of the isolates were deposited in the GenBank database

(http://www.ncbi.nlm.nih.gov/Genbank) with the accession numbers KJ775741, KJ775742,

KJ775743, KJ775744, KJ775745, KJ775746, KJ775747, KJ775748, KJ775749, KJ775750,

KJ775751, KJ775752, KJ775753, KJ775754, KJ775755, KJ775756.

3. 3.4. Microbial community analysis

In experiment 1, NMS ordination based on Bray-Curtis similarities revealed that the

microbiota associated with the mussel larvae seemed to group together according to

treatment (amorphous or crystalline) (Figure 3. 3). The analysis of similarities indeed showed

a significant global R value of 0.751 (p-value of 0.001) suggesting that similarity between

samples within a treatment is higher than the similarity between samples of different

treatments. Upon pairwise comparisons of treatments, however, no significant differences

could be determined due to the low number of replicates per treatment.

Figure 3. 3: NMS ordination based on Bray Curtis similarities for the bacterial communities associated with the larvae subjected to different feeding regimes in experiment 1. One replicate sample from treatment PHB-C 0.1 mg L-1 was left out of the analysis because it showed an abnormal low number of bands on the DGGE pattern

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Chapter 3 Application of PHB in mussel larviculture

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The NMS plot from experiment 2 suggests a considerable overlap in the composition of the

microbial communities associated with the larvae from the different treatments (Figure 3.

4). The non-significant differences between the microbial communities were confirmed by

the global R-value of only 0.027 and the p-value of 0.346.

Figure 3. 4: NMS ordination based on Bray Curtis similarities for the bacterial communities associated with the larvae subjected to different PHB concentrations in experiment 2.

3. 4. Discussion

Many studies focus on the development of sustainable strategies to improve the culture

performance and disease resistance of aquatically cultured animals. In this study, it was

shown for the first time that the supplementation of PHB can improve the survival of bivalve

larvae.

Even though mussel larvae are known filter feeders, a prerequisite for the experiments was

to determine whether PHB particles are taken up from the rearing water when offered to

the larvae. Defoirdt et al. (2007b) described the use of fluorescent labeling to visualize the

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Chapter 3 Application of PHB in mussel larviculture

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ingestion of PHB by Artemia franciscana larvae and it proved to be effective to demonstrate

the ingestion of PHB by mussel larvae within a time frame of two hours. The

supplementation of crystalline PHB to the culture water of the mussel larvae did not

significantly improve the survival, except for a temporary effect at the lowest concentration

until 14 days post hatching. This observation does not correspond with the results of

previous studies. A beneficial effect of PHB on survival was reported for Artemia franciscana

larvae (Defoirdt et al., 2007b), giant fresh water prawn larvae (Nhan et al., 2010, Thai et al.,

2014), and Chinese mitten crab larvae (Sui et al., 2012). The application method of PHB in

these studies (with the exception of the study on Artemia franciscana), however, was

different as PHB was administered either by addition to formulated feeds or by the

enrichment of live feed. This may partly explain the differences in observed effects. It is also

possible that the absence of improved survival is due to the mussel larvae not being able to

metabolize the crystalline PHB, despite their proven ability to ingest it, as D-stage mussel

larvae have an immature digestive system (Seafood-Industry-training-Organisation, 2006)

that may prevent them from efficiently metabolizing the crystalline PHB. In contrast, the

supplementation of amorphous PHB at a concentration of 1.0 mg L-1 did have a positive

effect on the larval survival. This may be due to the amorphous PHB being more

biodegradable than the crystalline PHB. Halet et al. (2007) made similar observations in case

of Artemia franciscana and ascribed this to the smaller size of the amorphous PHB and the

lower degradability of PHB in a crystalline structure. The crystalline particles in this study

have an average diameter of 30 µm, whereas the PHB containing bacteria are on average 1

µm in size. It cannot be overlooked, however, that the PHB is delivered to the mussel larvae

encapsulated within microbial biomass. This biomass may also have unintentionally

influenced the survival (for example by uncharacterized immunological stimulation). This

aspect needs to be further investigated.

The supplementation of PHB to the culture water of the mussel larvae did not result in a

faster growth nor in an improvement of the settlement success, not even for the PHB-A 1.0

mg.L-1 treatment that induced an increase in survival. De Schryver et al. (2010) in contrast

found that supplementing the diets of European sea bass with 2% or 5% crystalline PHB led

to a significant increase in fish weight gain as compared to the normal feed treatment (0%

PHB). Nhan et al. (2010) and Thai et al. (2014) demonstrated that the development of giant

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Chapter 3 Application of PHB in mussel larviculture

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freshwater prawn larvae fed with Artemia nauplii enriched with PHB in crystalline and

amorphous form, respectively, was significantly faster. Similarly, Sui et al. (2012) showed a

growth promoting effect of crystalline PHB on Chinese mitten crab larvae. As it was

previously suggested that PHB and/or its degradation products may be used as a fuel for

development and growth (Thai et al., 2014), this does not seem to be the case for mussel

larvae. Maybe the monomer of PHB, 3-hydroxybutyrate, is not used as a ketone body by

bivalves whereas this is the case for vertebrates and shrimp (Weltzien et al., 1999).

At this stage the exact mechanism by which the PHB polymer is broken down inside the

intestinal tract of animals is not known, i.e. whether it is mainly driven by physicochemical

processes or by biological activity of the host and/or microorganisms present in the gut

(Nhan et al., 2010). Defoirdt et al. (2007b) and (Liu et al., 2010) suggest that the metabolism

of PHB in brine shrimp larvae occurs at least partially by enzymatic activity of the host while

microbial activity may also play a role. This was based on the observation that the disease

resistance against the pathogen V. campbellii LMG21363 increased by the addition of PHB-

degrading bacteria, compared to the PHB treatment alone. To maximize the chance that

PHB-degrading isolates can persist and function in the specific ecological niche of the host

gastrointestinal tract, it is advisable to isolate them from this specific environment (Reid et

al., 2003). In the current study, all 16 (out of 22) PHB-degrading isolates seemed to belong to

the genus Pseudoalteromonas; isolate ARC4B5 showed the strongest PHB-degrading activity

and was thus selected for experiment 3. Its application, however, did not result in an

increase in survival and even seemed to lower the growth performance of the larvae. These

observations partly are in correspondence with the findings of Arlette et al. (2004), who

found a Pseudoalteromonas isolate that improved the survival of infected scallop (Pecten

maximus) larvae but also decreased their growth. Sandaa et al. (2008) describe a strain of

Pseudoalteromonas spp. as an opportunistic pathogen of P. maximus larvae. In our study, no

significant difference in survival between the control treatment and the Pseudoalteromonas

sp. treatment was observed.

It was earlier suggested that the beneficial effects associated with PHB may result from a

change in the gastrointestinal microbial community composition (De Schryver et al., 2011).

The differences in survival rates of mussel larvae in the different treatments could, however,

not be explained by differences in their respective microbial communities based on the

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Chapter 3 Application of PHB in mussel larviculture

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molecular analyses of the larvae associated microbial communities that were performed in

this study.

In conclusion, our results revealed that amorphous PHB, encapsulated in Ralstonia microbial

biomass, at a concentration of 1 mg L-1 had a positive effect on the survival of blue mussel

larvae. The total yield from egg to settled spat of 2 weeks old was 1.3 (experiment 2) to 2.4

(experiment 1 and 3) times higher than in the control treatment. Further research needs to

be performed to establish why amorphous PHB beneficially influences larval survival and not

larval growth. In addition, it is advised to determine if PHB may be used as an antimicrobial

compound to control pathogenic disease in blue mussel larvae like it does for other aquatic

species.

Acknowledgements

The Vietnam International Education Development (VIED) and Vlir-OI project (“Ensuring

bivalve seed supply in Central-Vietnam”, 2011-068) supported this work though a doctoral

grant to Nguyen Van Hung. Peter DS is supported as a post-doctoral research fellow by the

Research Foundation - Flanders (FWO, Belgium).

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Does Ralstonia eutropha, rich in poly-β

hydroxybutyrate (PHB), protect blue mussel

larvae against pathogenic vibrios?

Nguyen Van Hung1,2, Peter Bossier1, Nguyen Thi Xuan Hong1, Christine Ludeseve1, Linsey Garcia-Gonzalez3, Nancy Nevejan1, Peter De Schryver1

1 Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Rozier 44, B-9000 Gent, Belgium

2 Research Institute for Aquaculture no.3, 33 Dangtat, Nhatrang, Viet Nam

3 Vlaamse Instelling voor Technologisch Onderzoek (VITO), Boeretang 200, 2400 Mol, Belgium

CHAPTER 4: DOES RALSTONIA EUTROPHA, RICH IN POLY-Β HYDROXYBUTYRATE (PHB),

PROTECT BLUE MUSSEL LARVAE AGAINST PATHOGENIC VIBRIOS?

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Chapter 4 Ralstonia eutropha, rich PHB, protect blue mussel larvae

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Abstract

Disease outbreaks during larval rearing of commercial bivalve species are an important

impediment to the development of the mollusk aquaculture sector. Current research focuses

on finding alternatives to antibiotics and in this study the use of the natural polymer poly-β-

hydroxybutyrate (PHB) as a biological agent to control bacterial pathogens of blue mussel

(Mytilus edulis) larvae was investigated.

Blue mussel larvae were supplied with amorphous PHB (PHB-A: lyophilized Ralstonia

eutropha containing 75% PHB) at a concentration of 1 mg L-1 or 10 mg L-1 for 6 or 24 hours,

after which they were exposed to either the rifampicin resistant pathogen Vibrio splendidus

or Vibrio coralliilyticus at a concentration of 105 CFU mL-1 in well-plates containing 2 ml of

filtered-sterilized seawater enriched with 0.1% (v/v) of Luria-Bertani medium (salinity 35 g L-

1) and 10 mg L-1 rifampicin. Unchallenged larvae (with and without PHB-A) were used as

negative controls. LB35 (0.1%) medium was added to the water culture to stimulate the

growth of (only) the rifampicin-resistant pathogens. The survival of the larvae that were

PHB-A pretreated (1 mg L-1) for 6 h before the challenge was higher than when the pre-

treatment started 24 h before challenge. After 96h of pathogen exposure, the survival of

PHB-A treated mussel larvae was 1.41 and 1.76 fold higher than the survival of larvae not

treated with PHB-A when challenged with V. splendidus and V. coralliilyticus respectively. An

increase in PHB-A concentration from 1 to 10 mg L-1 significantly increased larval survival

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Chapter 4 Ralstonia eutropha, rich PHB, protect blue mussel larvae

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after 72 h and 48 h of a challenge with V. splendidus in the groups pretreated with PHB-A

for 6 h and 24 h respectively. The increased PHB-A concentration, however, did not lead to a

significant difference in the protection of mussel larvae against V. coralliilyticus A possible

mode of action includes the depolymerization of PHB by enzymatic or microbial activity into

the water-soluble short-chain fatty acid monomer, β-hydroxybutyrate (β-HB) that acts as a

microbial control agent. Therefore, growth inhibition of four concentrations of β-HB (1, 5, 25

and 125 mM) on the two pathogens was tested in vitro in LB35 medium, buffered at two

different pH values (pH7 and pH8). The highest concentration of 125 mM significantly

inhibited the pathogen growth in comparison to the lower levels. The effect of β-HB on the

virulence of the tested pathogenic Vibrios revealed a variable pattern of responses. The

highest β-HB concentration inhibited some virulence factors such as the production of the

lytic enzymes hemolysis, phospholipase but also stimulated the production of caseinase in V.

splendidus at pH7 and in V. coralliilyticus at pH8. β-HB had no effect on the production of

gelatinase and lipase or biofilm formation.

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Chapter 4 Ralstonia eutropha, rich PHB, protect blue mussel larvae

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4.1. Introduction

The production of many bivalve mollusk species depends on hatchery production of seed,

yet the intensive production of bivalve larvae is characterized by mass mortalities caused by

bacterial pathogens (Hada et al., 1984, Prado et al., 2010) In many cases, bacteria of the

genus Vibrio have been found to be the principle causative agents, with mortality often

exceeding 90% (Takahashi et al., 2000). Therefore, Vibriosis is considered a significant

disease threat for hatchery-reared larvae. To prevent infection by pathogenic Vibrio strains

many hatcheries adopt the use of antibiotics during larval culture (Nicolas et al., 1996,

Uriarte et al., 2001) although this is known to lead to the development of drug resistance in

pathogenic strains (Nicolas et al., 1996, Cabello, 2006, Fernandes Cardoso de Oliveira et al.,

2010). For this reason, the focus has shifted towards applying alternative biocontrol

techniques in bivalve rearing systems to prevent disease outbreaks. Probiotics, for example,

are considered a possible solution for controlling bacterial infections in bivalve larvae

(Douillet and Langdon, 1993, Kesarcodi-Watson et al., 2012). Alternatively, Defoirdt et al.

(2009) suggested that the polymer of the short-chain fatty acid (SCFA) β-hydroxybutyrate (ß-

HB), the well-known bacterial storage compound poly-β-hydroxybutyrate (PHB), could be

used to protect aquatic animals from pathogenic bacteria. Polyhydroxybutyric acid is an

important member of the family of polyhydroxyalkanoates (PHAs), and it can be degraded by

bacteria into the monomer β-HB (Kato et al., 1992, Patnaik, 2005). β-hydroxybutyrate acid is

known to have some antimicrobial, insecticidal and antiviral activities (Tokiwa and Ugwu,

2007). In addition, β-hydroxybutyrate is a known ketone body (Jendrossek and Handrick,

2002) that serves an important role in the energy metabolism of a large number of animals

(Nehlig, 2004). PHB can be supplied in either the crystalline (i.e. extracted from the bacterial

cell) or amorphous form (i.e. still contained within the bacterial cell) which after ingestion, is

thought to be depolymerized.Positive effects of PHB on growth and survival were shown for

a number of aquaculture species such as juvenile European sea bass (De Schryver et al.,

2010), juvenile Siberian sturgeon (Najdegerami et al., 2012), larval giant freshwater prawn

(Nhan et al., 2010, Thai et al., 2014), larval giant tiger prawn (Laranja et al., 2014), larval Nile

tilapia (Situmorang et al., 2016) and larval Chinese mitten crab (Sui et al., 2012). .

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Most recently, Hung et al. (2015) observed a 50 % increase in blue mussel larvae survival

when amorphous PHB (PHB-A) was supplied to the culture water at a concentration of 1 mg

L-1. However, no effect on larval growth or survival after metamorphosis was observed. It

was suggested that the increase in larval survival may have resulted from the antimicrobial

activity of the compound against opportunistic bacteria that were naturally present in the

water although that assumption was not verified.

This study investigated the effect of amorphous PHB in blue mussel larvae cultures in vivo

when challenged with the pathogenic bacteria Vibrio splendidus and Vibrio coralliilyticus.

Furthermore, the impact of βHB on the growth and some virulence factors of V. splendidus

or V. coralliilyticus was examined in vitro. The in vitro trials were carried out at pH7 and

pH8. Normal acidity of seawater is pH 8, but previous research demonstrated that β-HB was

more efficient as antimicrobial agent at a lower pH of 7 (Defoirdt et al., 2007b). In addition,

Defoirdt et al. (2009) hypothesized that a possible effect of PHB supplementation, is the

lowering of the pH value in the intestines of the host.

4. 2. Materials and Methods

4. 2.1. Bacterial strains and growth conditions

The pathogenic bacteria used in this study were V. coralliilyticus DO1 (Genbank accession

number EU358784) and V. splendidus 0529 (Genbank accession number EU358783) since

there are no pathogens described specifically for Mytilus edulis larvae to our knowledge. The

chosen pathogens were earlier reported to be pathogenic towards green shell mussel (Perna

canaliculus) larvae (Kesarcodi-Watson et al., 2009) and were kindly provided by Dr.

Kesarcodi-Watson (Cawthron Institue, New Zealand) for the current study.

Before use, the strains were made rifampicin resistant. Firstly, each bacterial strain stocked

in 40 % glycerol at - 80 °C was grown overnight at 18 °C in Luria-Bertani broth supplemented

with 35 g L-1 Instant Ocean sea salt (Aquarium Systems, Sarrebourg, France) (LB35). Further, a

volume of 500 µL was transferred into 5 mL of fresh LB35 containing rifampicin (which was

dissolved in DMSO) at a final concentration of 50 mg L-1. It took a few days before the

medium showed signs of growth. Afterward, 50 µl of this medium was plated on LB35 plates

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containing 50 mg L-1 rifampicin. A single colony was picked up from the plate, and the whole

procedure was repeated. The resistant strains were finally stored in 40 % glycerol at – 80 °C.

For use in the experiments, the rifampicin resistant bacteria were grown overnight in LB35

under constant agitation (130 rpm) at 18 °C, the temperature at which the challenges were

carried out. Cell densities were measured spectrophotometrically at 600 nm with an OD of 1

corresponding to about 1.2 x 109 colony forming units (CFU) mL-1. The final concentration of

pathogenic bacteria used in all challenge experiments was 105 CFU mL-1.

4. 2.2. Amorphous PHB (PHB-A)

The amorphous PHB consisted of lyophilized Ralstonia eutropha containing 75% PHB on cell

dry weight and was produced as described by Thai et al. (2014). The PHB-A was suspended

at 50 mg L-1 in FASW and sonicated for homogenization using an ultrasonic machine

(Branson 1200, USA). A final concentration of 1 mg L-1 and 10 mg L-1 PHB was utilized in the

challenge experiments.

4. 2.3. Experimental mussel larvae

Mature adult mussels were obtained from Roem van Yerseke (Yerseke, The Netherlands) the

day before spawning. The spawning and fertilization protocol were followed as described by

Hung et al. (2015). After fertilization, the development of the fertilized eggs was regularly

monitored. When the morula stage was reached in the majority of the embryos, they were

rinsed with filter autoclaved sea water (FASW) on a 30 μm sieve to wash away remaining

sperm. Next, the embryos were transferred to a glass bottle containing 2L of FASW at 18 °C

and a mixture of the antibiotics rifampicin, kanamycin, and ampicillin (10 mg L-1 each). After

48 hr, D-veliger larvae were obtained and washed at least five times with FASW to remove

the antibiotics. The larvae were re-suspended in fresh FASW, and the density was

determined. All manipulations were performed under laminar flow.

4. 2.4. Blue mussel larvae challenge tests

The protective effect of PHB-A against bacterial infection in blue mussel larvae was

evaluated in a number of challenge tests. The basis of each challenge test consisted of

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adding 100 mussel larvae into 24 inner wells (to avoid plate edge effects) of a sterile 48

well-plate (Tissue Culture Dish, Thermo Scientific). Each well contained 1 ml of FASW

supplemented with LB35 (0.1% v/v) and rifampicin (10 mg L-1). These 24 wells were divided

into 6 treatments with 4 replicate wells per treatment. This set-up was also applied in 24

well-plates for challenge tests. The plates were incubated at 18°C under a light regime 12D:

12L for all experiments. During the challenge test, the suspension in all wells was pipetted

very gently twice a day to re-suspend the PHB-A in the water column. Each challenge test

was repeated at least once. Only one sample experiment is shown here, since the results

were very consistent. Other results can be found in Appendix-C (see page on 189.)

Each challenge test consisted of following six treatments:

1) Non-treated larvae (“control”)

2) Larvae supplied with PHB-A immediately after stocking in the plates (“PHB-A”)

3) Larvae challenged with V. coralliilyticus at 6 or 24 h after stocking in the plates

(“V. coralliilyticus”)

4) Larvae challenged with V. splendidus at 6 or 24 h after stocking in the plates (“V.

splendidus”)

5) Larvae supplied with PHB-A immediately after stocking in the plates and

challenged 6 or 24 h later with V. coralliilyticus (“PHB-A + V. coralliilyticus”)

6) Larvae supplied with PHB-A immediately after stocking in the plates and

challenged 6 or 24 h later with V. splendidus (“PHB-A + V. splendidus”)

In each challenge test, the survival of the larvae was verified at 24h, 48h, 72h, and 96h after

introduction of the pathogens (except for control). At each of these time points, the larvae

of 3 well-plates were killed and stained with Lugol’s solution. Under the microscope, survival

was determined based on the presence of lugol coloured internal structures that included

the vellum, cilia, and stomach. Deterioration of organ structures and empty shells were

denoted as moribund or dead individuals (Figure 4.1). The conditions of the different

challenge tests are as explained in Table 4.1.

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Table 4. 1: Overview of the different blue mussel larvae challenge tests

Pathogens PHB concentration Period of PHB treatment

before challenge

Challenge test 1 V. coralliilyticus

V. splendidus

1 mg L-1 6 h

Challenge test 2 V. coralliilyticus

V. splendidus

1 mg L-1 24 h

Challenge test 3 V. coralliilyticus

V. splendidus

10 mg L-1 6 h

Challenge test 4 V. coralliilyticus

V. splendidus

10 mg L-1 24 h

Figure 4. 1: Blue mussel larvae after Lugol staining (x 400). Living larvae (A) are completely dark while partly (B) or entirely (C) empty shells are counted as dead larvae.

4. 2.5. Effect of β-HB on growth of V. coralliilyticus and V. splendidus

V. coralliilyticus and V. splendidus were grown overnight as described above and set at an

OD600 value of 1.0 in fresh LB35. The bacterial suspensions were inoculated (2% v/v) in 200 μl

LB35 containing different concentrations (0 mM, 1.0 mM, 5.0 mM, 25 mM and 125 mM) of β-

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Chapter 4 Ralstonia eutropha, rich PHB, protect blue mussel larvae

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HB (sodium β-hydroxybutyrate, Sigma-Aldrich, Germany) in a 96-well plate (n = 4 for each

pathogen-concentration combination). The assay was performed at pH 7 and pH 8 in the

presence of a 1M MOPS buffer. The plates were incubated in a spectrophotometer (Tecan I-

Control, Belgium) and the optical density of each well at 600 nm was measured every hour

for 24 h.

4. 2.6. Effect of β-HB on virulence factors of V. coralliilyticus and V. splendidus

For all virulence factors considered, filtered sterilized β-HB was added to the medium at

concentrations of 0 mM, 1 mM, 5 mM, 25 mM, or 125 mM. The pH of the medium was

consequently adjusted at 7 or 8 by the addition of 0.1 N HCl or 0.1 N NaOH.

All assays were performed in triplicate in at least two independent experiments with

consistent results.

4. 2.6.1. Swimming motility

A swimming motility assay was performed using LB35 plates with 2% agar ( Yang and Defoirdt

(2015)). The pathogens were spotted at the center of the plates, and the diameter of the

motility zones was measured after 24 h of incubation at 18 °C.

4. 2.6.2. Lytic enzyme production: caseinase, gelatinase, hemolysin, lipase and phospholipase

All assays were performed according to Natrah et al. (2011). The caseinase assay plates were

prepared by mixing equal volumes of autoclaved double strength LB35 agar and a 4%

skimmed milk powder suspension (Oxoid, UK), sterilized separately at 121 °C for 5 min.

Colony diameters and clearing zones were measured after 3 days of incubation at 28 °C.

Gelatinase assay plates were prepared by mixing 0.5% gelatin (Sigma-Aldrich) into LB35. After

incubation for 7 days, saturated ammonium sulfate (80%) in distilled water was poured over

the plates and after 2 min, the diameters of the clearing zones around the colonies were

measured.

Hemolytic assay plates were prepared by supplementing autoclaved LB35 agar with 5%

defibrinated sheep blood (Oxoid, Basingstoke, and Hampshire, UK). Colony diameters and

clearing zones were measured after 2 days of incubation at 18 °C.

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For the lipase and phospholipase assays, LB35 agar plates were supplemented with 1%

Tween 80 (Sigma-Aldrich) and 1% egg yolk emulsion (Sigma-Aldrich), respectively.The

diameter of the opalescent zones was measured after 3 days of incubation at 18 °C.

4. 2.6.3. Biofilm formation and exopolysaccharide production

A biofilm formation assay was conducted in 96 well plates as described by Li et al. (2014)

using LB35 broth. The exopolysaccharide (EPS) production assay was performed in a similar

way. For the quantification of exopolysaccharides, calcofluor white staining (Sigma-Aldrich)

was used as described by Brackman et al. (2008)

4. 2.7. Data analysis and statistics

Larval survival data in challenge tests were subjected to a logistic regression analysis using

GenStat (VSN International) version 16. The effects of varying concentrations of β-HB on the

virulence factors of the pathogenic bacteria were determined by first checking the

assumptions associated with ANOVA and in case any of the assumptions was violated, non-

parametric equivalents were considered. In effect, since the mean response was expected to

vary with a concentration level of β-HB, we were interested in testing trends in response to

increasing levels. Therefore, the Jonckheere Terpstra test for ordered alternatives was

applied. In situations where this test failed to indicate the presence of trends, the Kruskal-

Wallis test was used. All tests between the different concentrations of β-HB were tested at

the 5% significance level with the ANOVA Dunn-Bonferroni posthoc analysis to account for

multiplicity. All analyses were carried out using Statistical Package for the Social Sciences

version 23 (SPSS. Inc., Chicago. IL, USA) and R version 3.2.2 software packages.

4. 3. Results

4. 3.1. Survival of mussel larvae in challenge tests

The supplementation 6h before the introduction of the pathogens V. splendidus and

V.coralliilyticus of PHB-A at a concentration of 1 mg L-1, significantly increased larval survival

measured 96 h after the challenge (Figure 4.2, challenge 1). For all other larval

pretreatments in combination with V. coralliilyticus, no significant difference in survival with

the challenged control was observed.

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Chapter 4 Ralstonia eutropha, rich PHB, protect blue mussel larvae

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There was a significantly increased protection of mussel larvae against V. splendidus for 48 h

in the 24h pretreated PHB-A group in comparison to the non-pretreated challenged group.

(Figure 4.2, challenge 2). Compared to the challenged larvae that did not receive PHB-A, the

increase in PHB-A concentration from 1 to 10 mg L-1 significantly increased larval survival

after 72 h and 48 h of a challenge with V. splendidus in the groups pretreated with PHB-A for

6h and 24 h respectively. The increased PHB-A concentration, however, did not lead to a

significant difference in the protection of mussel larvae against V. coralliilyticus (Figure 4.2,

challenge 3 &4).

4. 3.2. Effect of β-HB on growth of V. coralliilyticus and V. splendidus

The impact of β-HB on the growth of the pathogens was species and pH dependent. The

growth of the two bacterial strains in LB35 was significantly inhibited at the highest β-HB

concentration of 125 mM at pH7 and pH8 except for the combination V.coralliilyticus at pH7

and pH8 (Figure 4.3).

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Chapter 4 Ralstonia eutropha, rich PHB, protect blue mussel larvae

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_ ................................................................................................................................................................................................................................................................................................................ !

i i~~·· re~ i 1

: • ''>+----i--,,, : I,

::::. 40 '+- · - · - · -~ ::::. 40 ' · ',, b

~ ~ 0 24 .. :2 .: ~ 2~ 0 24 .. - ~2- - -~ I Post exposure (h) Post exposure (h) I

·····+ ··· Control - · • · - V.splendidus ··· +···· Control - · • · - V.coralliilyticus

- PHB --•--V.splendidus + PHB - PHB - .... --V.coralliilyticus+ PHB ! Ch:~:nge test 2. 1 mg PHB L-

1; 24h

100

a I

'#. 80 ,,~~~>:~!, : : a '#. 80 '\ \, ~ a : : I ~ 60 f '':-',, b 1 60 ',I ~' 1,,

i!: 40 ' .: ... _ i!: 40 i'- ._ ....

~ 20 'a. >--- b b ~ 20 •• bb i 0 c -· b-:::~:~=~-=-lb 0 ·-...-~-=-~= ~= ~Q ____ b~b ' !

=::rr: :: .. ..:~_::~:::; .. "" :::rr~ o :,~:~:::::~ •• ". 1

;:~···~ ~ 1: \ : : : !,,!

i!: 40 r- ---- ..,~>--~ ~ 40 \

~ 20 c c --- : ~ 20 \ b. ! 0 0 -:..!?_ __ ~--..!>JL.

0

.. ....... Control

-PHB

24 48 72 96 Postexposure(h)

- .. - V.splendidus

- .... -- V.splendidus + PHB

Challenge test 4. 10 mg PHB L-1 ; 24h

100 ~: a a a • • • \ : ... a 80 ,_, a a a '#. \~ , b "iii 60 ~,, ' e. .::: '\, ........ b i!: 40 11.. '

~ ... ,, .. , ..... ..... ::::. c ""' ....... , .. ... ...... b "' 20 c ------- ~b bb .... ~:: .. -::-~::.~

0

0 24 48 72 96

Post exposure (h)

····•··· Cont rol ·-• -- V.s plendidus

- PHB - •- V.splendidus + PHB

0 24 48 72 96 Post exposure (h)

····•··· Cont rol -----V. coralliilyticus

-PHB --+- V. co ralliilyticus + PHB

100 ~: a a a • • • --~-' •a a a a

'#. 80 ---..t:> b \ '

"iii 60 ··.' .::: \' ',,, i!: 40 ' ' ::::. ',,_, b "' 20 ',, .. _- b

b ... _____ ::- .b ~

0 --- "'"-=----._--_.. 0 24 48 72 96

Post exposure (h) ·· ··• ··· Control --•-· V.coralliilyticus

- PHB - •- V.coralliilyticus + PHB I i "'·-··············································-····- ··········-·-···········-·················-···········-····················································-···········- -···········-·················-···········-···········-·····-·············································-1

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Figure 4. 2: Survival of blue mussel larvae challenged with V. coralliilyticus or V. splendidus at 105 CFU mL-1 under different experimental conditions. In challenge test 1 and 2, PHB was supplemented at a concentration of 1 mg L-1 6 h and 24 h respectively before the challenge, in challenge test 3 and 4, PHB was supplemented at a concentration of 10 mg L-1 6 h and 24h respectively before the challenge. Values are presented as means ± standard error (SE) of 3 replicate plates. The survival in both the control treatment (larvae only) and the PHB-A treatment without challenge was similar and therefore cannot be distinguished in the graphs. Values at the one-time point (just 24 h) that are marked with a different letter are significantly different (P < 0.05).

Figure 4. 3: Growth curves of V. splendidus and V. coralliilyticus (OD600) in LB35 at pH7 and pH8 with different concentrations of 0, 1, 5, 25, and 125 mM β-HB. Data are expressed as mean ± Standard Error (SE) of four replicates. An asterisk denotes a significant difference between the highest concentration of β-HB and the other concentrations. Error bars presented in the graph are often too small to be visible.

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4. 3.3. Effect of β-HB on virulence factors

4. 3.3.1. Swimming motility

Both Vibrio spp. used in this study exhibited swimming motility although patterns appeared

to differ between the two species (Table 4.2). The swimming motility of V. splendidus was

significantly higher with 25 mM β-HB at neutral pH, whereas no significant differences were

found between the different β-HB concentrations at pH8. The swimming motility of V.

coralliilyticus was only significantly affected at the highest concentration of 125 mM β-HB at

pH 7. At pH 8, no significant effects as compared to the control could be observed.

Table 4. 2: Swimming motility zone of V. splendidus and V.coralliilyticus after 24h incubation

on soft agar at pH7 and pH8 with different β-HB concentrations.

V. splendidus V. coralliilyticus

β-HB

(mM)

pH7 pH8 pH7 pH8

Motility zone (mm) Motility zone (mm) Motility zone (mm) Motility zone (mm)

0 40.0 ± 0.7a 33.5 ± 0.5a 45.3 ± 0.3b 47.5 ± 1.0ab

1 38.0 ± 2.0a 30.0 ± 0.7a 44.8 ± 0.3ab 45.0 ± 0.0a

5 46.8 ± 1.0ab 32.5 ± 1.0a 46.0 ± 0.7b 45.8 ± 0.8ab

25 57.3 ± 3.5b 31.8 ± 2.3a 41.3 ± 0.5ab 49.8 ± 0.9b

125 43.8 ± 0.9ab 30.8 ± 1.7a 35.3 ± 1.0a 46.0 ± 0.7ab

Data are expressed as mean ± Standard Error (SE) of four replicates. Values in the same column with different letters indicate significant differences in motility zones between β-hydroxybutyrate concentrations (p<0.05). All pairwise comparisons were done using the Dunn-Bonferroni method.

4. 3.3.2. Lytic enzymes activity

The activity of different lytic enzymes (caseinase, gelatinase, lipase, hemolysin, and

phospholipase) of V. splendidus and V. coralliilyticus upon supplementation of varying

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concentrations of β-hydroxybutyrate at different pH levels is shown both Vibrio strains

tested positive for all virulence factors in the absence of β-HB. Overall, the colonies and

clearing zones on the caseinase assay plates were noticeably smaller at the highest

concentration of β-HB. The ratio between the clearing zone and colony diameter was

significantly higher at 125 mM β-HB as compared to 0 mM (except for V. splendidus at pH8).

A significantly stronger gelatinase activity was observed at 5 mM β-HB for V. coralliilyticus at

pH8. For the other concentrations of β-HB, there were no significant differences in the

control treatment of 0 mM β-HB for both pathogens at both pH values (Table 4. 2).

There was no significant effect of β-HB on lipase at any of the β-HB concentrations for both

pathogens. The ratio in V. splendidus was bigger than in V. coralliilyticus, whereas the

colonies in V. splendidus were smaller than in V. coralliilyticus.

On the contrary, an effect of β-HB on the hemolytic activity was observed in both pathogens

and both pH levels. The highest level of β-HB always showed a significant difference as

compared to the control treatment (0 mM βHB), being higher for V. splendidus at pH 7 and

lower in all other cases.

While phospholipase activity was significantly decreased at 125 mM for V. splendidus at both

pH7 and pH8, there was no significant effect for V. coralliilyticus.

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Table 4. 3: The activity of different lytic enzymes (caseinase, gelatinase, lipase, hemolysin,

and phospholipase) of V. splendidus and V. coralliilyticus upon supplementation of varying

concentrations of β-hydroxybutyrate at various pH levels

β-HB

(mM)

pH7 pH8

Colony

diameter

(mm)

Clearing

zone

(mm)

Ratio Colony

diameter

(mm)

Clearing

zone

(mm)

Ratio

Caseinase/ V.splendidus

0 18.0 ± 0.0 22.0 ± 0.0 1.2 ± 0.0a 25.5 ± 1.0 29.5 ± 1.7 1.2 ± 0.0ab

1 20.0 ± 0.7 24.5 ± 1.2 1.2 ± 0.0a 26.0 ± 1.1 31.3 ± 1.8 1.2 ± 0.0ab

5 20.0 ± 0.4 23.5 ± 0.6 1.2 ± 0.0a 25.5 ± 0.5 29.3 ± 0.8 1.1 ± 0.0a

25 17.3 ± 0.9 21.3 ± 1.3 1.2 ± 0.0a 19.0 ± 0.0 24.0 ± 0.0 1.3 ± 0.0b

125 5.0 ± 0.0 10.0 ± 0.0 2.0 ± 0.0b 18.5 ± 0.3 21.8 ± 0.3 1.2 ± 0.0ab

Caseinase / V.coralliilyticus

0 19.0 ± 0.6 24.3 ± 0.3 1.3 ± 0.0a 24.5 ± 0.3 29.3 ± 0.5 1.2 ± 0.0a

1 21.8 ± 0.3 26.0 ± 0.4 1.2 ± 0.0a 26.0 ± 0.0 30.0 ± 0.0 1.2 ± 0.0a

5 18.5 ± 0.9 21.0 ± 1.0 1.1 ± 0.0a 25.3 ± 0.3 28.8 ± 0.8 1.1 ± 0.0a

25 17.0 ± 0.4 20.8 ± 0.3 1.2 ± 0.0a 22.8 ± 0.9 24.8 ± 0.3 1.1 ± 0.0a

125 5.0 ± 0.0 10.0 ± 0.0 2.0 ± 0.0b 17.0 ± 0.0 23.0 ± 0.0 1.4 ± 0.0b

Gelatinase/V.splendidus

0 18.5 ± 1.6 34.3 ± 0.5 1.9 ± 0.1ab 19.0 ± 0.6 35.3 ± 0.3 1.9 ± 0.1ab

1 15.3 ± 0.5 30.5 ± 1.0 2.0 ± 0.0b 15.0 ± 1.3 32.5 ± 1.0 2.2 ± 0.1b

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5 25.3 ± 1.9 35.0 ± 3.1 1.4 ± 0.1a 20.0 ± 0.0 35.0 ± 0.0 1.8 ± 0.0ab

25 22.8 ± 1.0 34.0 ± 0.7 1.5 ± 0.0ab 22.3 ± 1.0 35.5 ± 0.5 1.6 ± 0.1a

125 14.5 ± 1.2 26.0 ± 0.8 1.8 ± 0.1ab 16.0 ± 0.0 30.0 ± 0.0 1.9 ± 0.0ab

Gelatinase/ V.coralliilyticus

0 21.5 ± 1.2 33.5 ± 0.5 1.6 ± 0.1ab 19.5 ± 0.3 33.0 ± 0.6 1.7 ± 0.0a

1 13.8 ± 1.3 29.0 ± 0.6 2.1 ± 0.2b 16.0 ± 0.7 33.0 ± 0.6 2.1 ± 0.1ab

5 19.8 ± 1.3 32.8 ± 1.1 1.7 ± 0.1ab 14.0 ± 0.0 33.3 ± 0.5 2.4 ± 0.0b

25 27.8 ± 3.0 38.0 ± 0.0 1.4 ± 0.2a 16.0 ± 2.0 33.3 ± 0.8 2.1 ± 0.3ab

125 15.3 ± 0.9 27.3 ± 0.5 1.8 ± 0.1ab 15.8 ± 0.5 32.0 ± 0.0 2.0 ± 0.1ab

Lipase/ V.splendidus

0 7.6 ± 0.2 16.5 ± 0.3 2.2 ± 0.1a 7.5 ± 0.3 14.5 ± 0.3 1.9 ± 0.0ab

1 6.8 ± 0.6 13.5 ± 0.6 2.0 ± 0.1a 7.5 ± 0.5 12.5 ± 0.6 1.7 ± 0.1a

5 7.8 ± 0.3 15.8 ± 0.5 2.0 ± 0.1a 8.1 ± 0.1 16.3 ± 0.3 2.0 ± 0.0b

25 8.3 ± 0.3 16.3 ± 0.5 2.0 ± 0.1a 8.0 ± 0.4 16.0 ± 0.8 2.0 ± 0.0b

125 8.3 ± 0.1 15.5 ± 0.3 1.9 ± 0.0a 7.8 ± 0.3 14.5 ± 0.3 1.9 ± 0.1ab

Lipase/ V.coralliilyticus

0 15.5 ± 0.9 19.5 ± 0.5 1.4 ± 0.1a 14.8 ± 0.3 19.8 ± 0.3 1.3 ± 0.0a

1 14.0 ± 0.6 18.5 ± 0.6 1.3 ± 0.0a 15.0 ± 0.4 19.0 ± 0.4 1.3 ± 0.0a

5 13.8 ± 1.3 18.8 ± 0.9 1.4 ± 0.1a 12.8 ± 0.5 20.3 ± 0.9 1.6 ± 0.1a

25 17.3 ± 0.8 21.3 ± 0.6 1.2 ± 0.0a 14.5 ± 0.5 19.3 ± 0.8 1.3 ± 0.0a

125 13.8 ± 0.5 18.5 ± 0.3 1.3 ± 0.0a 13.8 ± 0.5 18.3 ± 0.6 1.3 ± 0.0a

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Hemolytic/ V.splendidus

0 18.8 ± 0.6 15.5 ± 0.6 0.8 ± 0.1a 11.3 ± 1.1 19.5 ± 1.0 1.8 ± 0.1a

1 9.3 ± 0.1 12.3 ± 0.1 1.3 ± 0.0ab 10.5 ± 0.3 12.5 ± 0.3 1.2 ± 0.0bc

5 18.3 ± 1.1 16.0 ± 0.3 0.9 ± 0.2ab 14.8 ± 2.0 13.5 ± 0.9 0.9 ± 0.1c

25 11.5 ± 0.7 13.8 ± 0.4 1.2 ± 0.1ab 9.0 ± 0.4 12.5 ± 0.3 1.4 ± 0.1b

125 7.0 ± 0.0 10.5 ± 0.1 1.5 ± 0.0b 9.0 ± 0.0 12.3 ± 0.3 1.4 ± 0.0b

Hemolytic/ V.coralliilyticus

0 9.0 ± 0.0 15.5 ± 0.3 1.7 ± 0.0a 9.0 ± 0.0 18.0 ± 0.0 2.0 ± 0.0a

1 9.5 ± 0.3 11.8 ± 0.3 1.2 ± 0.0ab 9.0 ± 0.0 12.0 ± 0.0 1.3 ± 0.0ab

5 10.0 ± 0.0 12.0 ± 0.0 1.2 ± 0.0ab 9.0 ± 0.0 12.0 ± 0.0 1.3 ± 0.0ab

25 10.0 ± 0.4 11.8 ± 0.3 1.2 ± 0.0ab 9.5 ± 0.3 12.0 ± 0.0 1.3 ± 0.0ab

125 10.8 ± 0.3 13.0 ± 0.0 1.2 ± 0.0b 11.5 ± 0.5 13.3 ± 0.5 1.2 ± 0.0b

Phospholipase/ V.splendidus

0 10.0 ± 0.0 14.5 ± 0.3 1.5 ± 0.0a 11.0 ± 0.6 14.3 ± 0.3 1.3 ± 0.1a

1 12.0 ± 0.0 15.8 ± 0.3 1.3 ± 0.0ab 13.5 ± 0.5 15.5 ± 0.5 1.1 ± 0.0ab

5 10.0 ± 0.3 13.0 ± 0.0 1.3 ± 0.0ab 12.8 ± 0.5 15.8 ± 0.5 1.2 ± 0.0a

25 11.8 ± 0.3 15.8 ± 0.3 1.3 ± 0.0ab 13.0 ± 0.0 15.3 ± 0.3 1.2 ± 0.0a

125 11.0 ± 0.3 13.5 ± 0.3 1.2 ± 0.0b 10.5 ± 0.3 11.5 ± 0.3 1.1 ± 0.0b

Phospholipase/ V.coralliilyticus

0 13.8 ± 0.3 18.8 ± 0.8 1.4 0.1ab 15.3 ± 0.5 18.5 ± 0.5 1.2 0.0ab

1 14.5 ± 0.3 21.0 ± 0.0 1.4 0.0ab 13.0 ± 0.0 16.3 ± 0.3 1.3 0.0a

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5 12.5 ± 0.5 17.0 ± 0.0 1.4 0.1ab 12.0 ± 0.0 16.0 ± 0.0 1.3 0.0a

25 13.0 ± 0.0 15.0 ± 0.0 1.2 0.0b 12.0 ± 0.0 14.0 ± 0.0 1.2 0.0ab

125 13.3 ± 0.3 15.5 ± 0.3 1.2 0.0b 12.0 ± 0.0 13.3 ± 0.5 1.1 0.0b

Data are expressed as mean ± standard error (SE) of four replicates. Ratio values for each virulence factor-species combination in the same column indicated with a different letter are significantly different (P < 0.05). All pair wise comparisons were done using Dunn-Bonferroni method. The ratio of colony diameter and clearing zone data were transformed (normality and homogeneity) to satisfy assumptions of ANOVA.

There was no significant effect of β-HB on biofilm formation for both pathogens at both pH

values compared to the control treatment (Figure 4. 4 a & b). The exopolysaccharide

production more or less reflected the tendencies observed for the biofilm formation (Figure

4. 4 c & d). A significant increase was found though at 5 mM and pH8 in the case of V.

splendidus (Figure 4. 4 c).

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Figure 4. 4: Biofilm formation (a & b) and exopolysaccharide production (c & d) of V. splendidus and V.coralliilyticus after 4 days incubation at 18°C in different ß-HB concentrations at pH7 and pH8 (average ± standard error of four replicates). Different letters indicate significant differences between β-HB levels (a,b for comparison at pH7; x,y for comparison at pH8). All pairwise comparisons were done using Dunn-Bonferroni method.

4. 4. Discussion

The results obtained in the present work demonstrated that the administration of PHB-A

had a positive effect in vivo on the survival of blue mussel larvae when it was administered 6

h before the challenge with V. splendidus or V. coralliilyticus. To our knowledge, this is the

first time that PHB-A has been tested as a protective compound for blue mussel larvae in

controlled challenge tests. It was also confirmed that the two tested vibrios are also virulent

for blue mussel larvae.

Currently, the exact mode of action by which PHB induces its protective effects is still not

clear. In general, it is assumed that the compound is bio-converted to its monomer β-HB

upon ingestion and that this compound then acts as an antimicrobial agent (Defoirdt et al.,

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2009), provides direct energy to the host (Fernández, 2001) or acts as an immunostimulant

(Baruah et al., 2015). In previous studies, PHB-A has been shown to significantly increase the

survival of Artemia when challenged with V. campbellii under both gnotobiotic and

conventional conditions (Halet et al., 2007, Defoirdt et al., 2007b). Consistent with a

previous report on Artemia (Defoirdt et al., 2007b), our results provided evidence that the

addition of amorphous PHB to the mussel larvae culture water significantly improves the

protection of larvae against both pathogens V. splendidus and V. coralliilyticus, although

there were major differences in the effective PHB-A dose between the two studies. In the

Defoirdt et al. (2007b) study, maximum and complete protection against V. campbellii was

obtained at a PHB-A concentration of 1000 mg L-1 while the lower PHB-A concentration of

100 mg L-1 still provided significant protection. In contrast, in the present study, an increase

in PHB-A concentration from 1 mg to 10 mg PHB-A mL-1 did not improve the larval survival.

This result supports our previous in vivo study (Hung et al., 2015) where the beneficial

effects of PHB-A in the culture of blue mussel larvae were optimal at 1 mg L-1, with inferior

results obtained at the higher concentration of 10 mg L-1.

Another interesting observation that was noted in this study was that the time window

between treatment and challenge influenced the outcome of the challenge. When the

pretreatment with PHB-A was administered 24h before the challenge, no positive effect on

survival was observed. In this case, it is possible that the PHB-A had been rapidly degraded

by the natural enzymes or resident PHB-degraders in the digestive system of the larvae.

Therefore, PHB-A treated mussel larvae failed to accumulate enough PHB concentration

when pretreated 24h before the challenge (in contrast to the 6 h pre-treatment).

In order to determine the effect of the monomer β-HB on the pathogens in this study, an

effort was made to measure the concentration of β-HB in the mussel larvae themselves.

However, due to the small dimensions of the mussel larvae, values remained below the

detection limit. In another study, however, it was found that the β-HB concentration in

Artemia nauplii supplied with PHB ranged between 1 and 8 mM (De Schryver et al.,

unpublished data). Based on these values, it was decided to determine the effect of β-HB on

the growth and virulence factor activity of the pathogens at concentrations of 1 and 5 mM,

this being a realistic estimation of the PHB concentration in the gut of blue mussel larvae

treated with PHB. In addition, higher PHB concentrations of 25 and 125 mM were also

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included to provide a better insight on the effect of β-HB. The effect of these concentrations

were verified at two pH values. Indeed, pH of the environment is a very important factor

since the growth-inhibitory effect of β-HB was earlier shown to decrease with increasing pH.

At pH 5, the growth of V. campbellii was completely inhibited; at pH 6, growth was delayed

and at pH 7, no inhibition could be observed (Defoirdt et al., 2006b). The pH-dependency

can be explained by the fact that fatty acids can pass the cell membrane only in their

undissociated form, which will be more pronounced at lower pH (see the Henderson-

Hasselbach equation (Sun et al., 1998)).

From the in vitro assays, it became clear that the pathogens’ growth was not inhibited at the

lower concentrations of β-HB. Only a concentration of 125 mM β-HB significantly inhibited

the growth of both pathogens V. splendidus and V. coralliilyticus at neutral pH (except for V.

coralliilyticus at alkaline pH). This indicates that the beneficial effect of PHB on larval survival

was unlikely the consequence of β-HB reducing the growth of the pathogens as it is exactly

these lower concentrations that can be expected in the gastrointestinal environment of the

blue mussel larvae. For this reason, it was determined if there could have been an effect of

β-HB on the virulence factors of the pathogens.

A proper evaluation of the in vitro test requires a better understanding of the general and

specific stress response capabilities of pathogens. The degree of pathogenicity, also termed

virulence depends on the expression of virulence factors, i.e. gene products that enable the

pathogen to infect and damage the host, including proteins involved in motility and

adhesion of the pathogen to the host, protection from host defence mechanism and host

tissue degradation, iron acquisition and toxins (Chen et al., 2005, Defoirdt, 2014).

Bacterial motility is now considered as an important virulence factor for many pathogens. It

is essential for pathogenic bacteria during the initial phases of infection as it helps them to

overcome repulsive forces between the bacterial cell and the host tissues and hence,

facilitates attachment to the host (McCarter, 2001). Our results indicated that the effect of

β-HB on the swimming motility of pathogens depended both on the species and

concentration. V. splendidus’ swimming activity significantly increased at 25 mM of β-HB

while V. coralliilyticus’ motility was significantly decreased at 125 mM (pH7).

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Biofilm formation is an ancient and integral component of the prokaryotic life cycle and is a

critical factor for survival in diverse environments (Hall-Stoodley et al., 2004). This process in

Vibrios depends on specific genes (flagella, pili, and exopolysaccharide biosynthesis) and

regulatory processes (Yildiz and Visick, 2009). In the present study, β-HB did not exert any

influence on the biofilm formation of the two Vibrio spp. investigated. Irrespective of the

presence or absence of β-HB, biofilm formation seemed to be very low in both Vibrio spp.

For Vibrio fischeri, Chavez-Dozal and Nishiguchi (2011) found biofilm formation at an OD562

to be 0.83 and 1.10 at 20 and 25°C, respectively, whereas our results showed minimum-

maximum values of 0.04-0.10 and 0.04-0.15 in V. splendidus and V. coralliilyticus,

respectively. The small biofilm formation observed in the present study could be related to

the sub-optimal incubation temperature used (18 °C). This context applies in particular for V.

coralliilyticus. Several studies have identified V. coralliilyticus as a mesophilic bacterium with

its virulence factors upregulated at elevated temperatures (Kimes et al., 2012, Frydenborg et

al., 2014). This is not applicable however for V. splendidus that grows at low temperatures,

being psychrotroph (Pujalte et al., 1999).

Extracellular proteases have been identified to play a significant role in virulence and

pathogenicity of many bacteria (Cai et al., 2007). Defoirdt (2014) found that lytic enzymes

produced by aquaculture pathogens include hemolysins and proteases. Haemolysin is one of

the virulence factors of pathogens described for shrimp, fish (Liu et al., 1996, Sun et al.,

2007) and bivalve mollusks (Nottage and Birkbeck, 1990). Secreted phospholipases are

thought to function in phosphate acquisition, carbon source acquisition, and in some cases

as virulence factors for pathogenic species (Schmiel and Miller, 1999). The results of the

assays indicated that β-HB significantly affected the virulence factors only in a limited

number of cases (Table 4. 4).

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Table 4. 4: Summary of the effects of β-HB on selected virulence factors of mussel larvae

Vibrio pathogens

V. splendidus V. coralliilyticus

pH 7 pH 8 pH 7 pH 8

Caseinase 125 mM No effect No effect 125 mM

Gelatinase No effect No effect No effect No effect

Lipase No effect No effect No effect No effect

Hemolysin 125 mM 1, 5, 25 ,125 mM 125 mM 125 mM

Phospholipase 125 mM 125 mM No effect No effect

Swimming motility 25 mM No effect 125 mM No effect

Biofilm formation No effect No effect No effect No effect

Exopolysaccharide No effect 5 mM No effect No effect

Focusing on the concentrations that can be considered to be relevant to the gastrointestinal

environment of blue mussel larvae (range 1- 25mM), only one beneficial effect of β-HB for

the host can be reported: at a β-HB concentration of 1, 5 and 25mM, the hemolytic enzyme

activity in V. splendidus is inhibited at pH8. The hypothesis is that PHB may affect the

virulence factor activity of the pathogens under investigation and as such protect the blue

mussel larvae from infection, is here with rejected. It is likely that the protective effect of

PHB on blue mussel larvae involves other mechanisms such as immunostimulation (Baruah

et al., 2015) or the direct provision of energy (De Schryver et al., 2010).

In conclusion, the present study indicated that PHB-A can partly protect mussel larvae

against specific pathogenic bacteria and that the effect of PHB-A on larval survival in the

challenge experiments depended on both PHB-A concentration and exposure time. In

general, pretreatment of mussel larvae with PHB-A at a concentration of 1 mg L-1 resulted in

better larval survival when administered 6h before the challenge, compared to

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administration of PHB-A 24h before the challenge. Larvae with the 6h pretreatment showed

a 55% and 25% increase in survival after exposure to the pathogens V. splendidus and V.

coralliilyticus, respectively, as compared to challenged larvae that were not treated with

PHB-A. Increasing the pretreatment from 1 to 10 mg PHB-A L-1 did not improve the

protection of mussel larvae against the challenge unless treatment was suspended PHB-A at

6h challenged with V. splendidus. This result is in accordance with the previous mussel larvae

experiments confirming that PHB-A of 1 mg L-1 is an optimal concentration in mussel larvae

culture (Hung et al., 2015).

The in vitro experiments suggested that β-HB affected the virulence factor activity of the

pathogens rather than growth, although the responses didn’t follow a clear pattern. The

highest β-HB concentration of 125 mM inhibited some virulence factors of the pathogenic

bacteria such as hemolysis, phospholipase, but also stimulated caseinase production. The

lowest β-HB concentrations inhibited a hemolytic enzyme (1 mM) in contrast, EPS was

stimulated at (5mM) β-HB in V. splendidus at pH8. There was no effect on the production of

the lytic enzymes gelatinase and lipase and the formation of biofilm were not affected by the

presence of β-HB at any concentration. The in vitro and in vivo tests of this study confirmed

that both Vibrio coralliilyticus and V. splendidus are also virulent for blue mussel larvae.

Acknowledgements

The Vietnamese International Education Development Agency (VIED), the Vlir-OI project

(“Ensuring bivalve seed supply in Central-Vietnam” no2011-068) supported this work

through a doctoral grant to the first author. The Research Foundation - Flanders (FWO,

Belgium), supported Peter DS as a post-doctoral research fellow. This research was also

financed by Ghent University through the Special Research Fund “Host-microbial

interactions in aquatic production” project (BOF12/GOA/022). We wish to thank Dr.

Kesarcodi-Watson (Cawthron Institue, New Zealand) kindly provided the bacterial strain for

this research and Dr. Abatih Emmanuel (FIRE, University of Ghent) for statistical guidance.

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Ralstonia eutropha, containing high poly-β-

hydroxybutyrate levels (PHB-A), regulates the

immune response in mussel larvae challenged with

Vibrio coralliilyticus

Nguyen Van Hung1,2, Nancy Nevejan1, Peter De Schryver1, Nguyen Viet Dung1 and Peter Bossier1

1Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Rozier 44, B-9000 Gent, Belgium

2Research Institute for Aquaculture no.3, 33 Dangtat, Nhatrang, Viet Nam

CHAPTER 5: RALSTONIA EUTROPHA, CONTAINING HIGH POLY-Β-HYDROXYBUTYRATE LEVELS

(PHB-A), REGULATES THE IMMUNE RESPONSE IN MUSSEL LARVAE CHALLENGED WITH

VIBRIO CORALLIILYTICUS

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Abstract

Marine invertebrates rely solely on innate immune mechanisms that include both humoral

and cellular responses. Antimicrobial peptides (AMPs), lysozyme and phenoloxidase activity,

are important components of the innate immune defense system in marine invertebrates.

They provide an immediate and rapid response to invading microorganisms. The impact of

amorphous poly-β-hydroxybutyrate (PHB-A) (1mg PHB-A L-1) on gene expression of the

AMPs mytimycin, mytilinB, defensin and the hydrolytic enzyme lysozyme in infected blue

mussel larvae was investigated during “in vivo” challenge tests with Vibrio coralliilyticus (105

CFU mL-1). RNAs were isolated from mussel larvae tissue, and AMPs were quantified by q-

PCR using the 18SrRNA gene as a housekeeping gene. Our data demonstrated that AMPs

genes had a tendency to be upregulated in challenged mussel larvae, and the strongest

expression was observed from 24h post-exposure onwards. The presence of both PHB-A and

the pathogen stimulated the APMs gene expression however; no significant differences were

noticed between treatments and exposure time. Looking at the phenoloxidase activity in the

infected mussels, it was observed that the addition of PHB-A induced increased activity.

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5.1. Introduction

In common with other invertebrates, bivalves rely solely on innate immunity, which by

definition lacks adaptive characteristics, to combat against invading pathogens.

Antimicrobial peptides appear to be one of the actors in innate immunity that have been

conserved during evolution, although their involvement in anti-infectious processes is

different according to species, cell type, and tissue (Mitta et al., 2000b). Many antimicrobial

peptides are located in epithelia (Schröder, 1999) where they prevent invasion by pathogens

while others may be especially abundant in circulating cells. They are recognized to be a

major component of the innate immune defense system in bivalve mollusks as well (Cellura

et al., 2007).

The innate immunity in mollusks is not well understood. Nevertheless, pathogen recognition

receptors (PRRs) have been identified in some species (Araya et al., 2010). Once the

presence of microbes is detected, these intruders are phagocytosed and destroyed by toxic

radicals, lysozyme and antimicrobial peptides, which are involved in bacterial killing by

destabilizing their membrane permeability (Hancock and Rozek, 2002). Jenssen et al. (2006)

described in detail the interaction process between the peptide and target cell. It is thought

to occur through electrostatic bonding between the cationic peptide and the negatively

charged components present on the bacterial outer envelope, such as phosphate groups

within the lipopolysaccharides of Gram-negative bacteria or lipoteichoic acids present on the

surface of Gram-positive bacteria.

In mussels, four AMPs (defensin, mytilin, myticin and mytimycin) which play a key role in the

immune defense were identified and characterized (Mitta et al., 2000b). Different mussel

species have various AMPs genes, e.g., myticin appears in Mytilus galloprovincialis only,

whereas mytimycin occurs in M. edulis only (Tincu and Taylor, 2004). Some of them have a

wide spectrum of action while others are target-specific. For instance, mytimycin is

described as being strictly anti-fungal, mytilin acts extracellularly, whereas myticin can act

either intracellularly (during phagocytosis) or extracellularly (Mitta et al., 2000b). Mitta et al.

(2000b) also reported that defensin producing granulocytes are concentrated in the

intestinal epithelia, whereas mytilin and myticin expressing granulocytes are well

represented in gills suggesting that the type of AMP may dictate granulocyte allocation in

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different organs in mussels. M. galloprovincialis defensin 1 (MGD1) is an original member of

the arthropod defensin family due to the presence of 2 extra-cysteines and one modified

amino acid (Mitta et al., 1999b). Defensin, mytilin and myticin were determined in vitro to

have antimicrobial activity. Differences in expression of these AMPs genes were recorded

when mussel M. galloprovincialis adults were challenged with various factors such as

bacterial infections or by heat shock (Cellura et al., 2007). To our knowledge, nothing is

known about the genes that code for these antimicrobial peptides in blue mussel M. edulis

larvae or about the regulation of their expression. Besides the AMP gene expression,

another critical component of the immune system of bivalves namely phenoloxidase activity

(PO) was examined. As the product of a complex cascade of reactions, PO is generated from

proPO through a limited proteolysis by a proPO activating enzyme (Asokan et al., 1997), and

is involved in melanization, encapsulation, wound healing, phagocytosis, and pathogen

extermination (Bai et al., 1997, Munoz et al., 2006). The soluble form of PO is always

involved in humoral immunity while the cellular PO that binds to the surface of hemocytes,

is more associated with cell-mediated immunity (Hellio et al., 2007). Bacterial infection can

cause a significant increase in PO in the hemolymph of the bivalves Crassostrea madrasensis

and Chlamys farrization (Cong et al., 2008).

The compound poly-ß-hydroxybutyrate (PHB), a polymer of the short-chain-fatty acid ß-

hydroxybutyrate, was proven to protect experimental animals against a variety of bacterial

diseases, including vibriosis in farmed aquatic animals, albeit through undefined mechanisms

(Baruah et al., 2015). Recent research demonstrated that amorphous PHB-A, namely

Ralstonia cells containing more than 75% PHB on dry weight basis, in particular increases the

survival of mussel larvae (M. edulis) (Hung et al., 2015) and protects them during in vivo

challenge tests with V. coralliilyticus and V. splendidus in the previous study (chapter 4).

This study focuses on the detection and regulation of the three AMPs mytimycin, mytilinB,

defensin and the hydrolytic lysozyme in M. edulis larvae. On a second level the impact of

PHB-A on the expression of these genes and on PO activity regulation was investigated

during challenge tests with the pathogen V.coralliilyticus.

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5. 2. Materials and methods

5.2.1. Bacterial strains and growth conditions

Vibrio coralliilyticus is a Gram-negative marine bacterium isolated at the Glen Haven

Aquaculture Centre (New Zealand) during 2004 and 2005. This selected strain was reported

as a pathogenic Vibrio for bivalve larvae culture (Kesarcodi-Watson et al., 2009b). V.

coralliilyticus used in the experiments was mutated to a rifampicin resistant strain (in

previous study - chapter 4). Before use, 10 µl of the stored cultures (in 40% glycerol at -80

°C) were plated on Luria- Bertani plates to which 35g L-1 of Instant Ocean® (LB35 agar) was

added and incubated for 24 h at 17 °C. Single colonies were picked from the plates and

cultured overnight in fresh LB35 at 17 °C under constant agitation (150 min-1) before each

experiment.

5.2.2. Spawning procedure and larvae handling

Mature blue mussels (Mytilus edulis) were transported from the mussel producer Roem van

Yerseke, in The Netherlands to the Laboratory of Aquaculture and Artemia Reference

Center, Ghent University, Belgium, under cooled conditions. The protocol for mussel

spawning and fertilization was followed as described in Hung et al. (2015). The development

of the fertilized eggs was regularly monitored. When the morula stage was reached in the

majority of the embryos, the embryo solution was sieved through a 30 μm sieve. The

remaining sperm was washed away with 0.22 μm filtered and autoclaved seawater (FASW).

After this washing step, the embryos were transferred to a 2L glass bottle containing fresh

FASW at 18 °C and a mixture of antibiotics (10 mg l-1) to minimize bacterial interference (in

previous study-chapter 4). After 48h, the straight-hinged D-larvae were washed at least five

times with FASW to remove every trace of antibiotics and then re-suspended in fresh FASW.

All manipulations were performed under a laminar flow hood to avoid contamination before

the start of the challenge tests.

5.2.3. Challenge tests

The first experiment was carried out in 48-well tissue culture dishes (TCD), and the second,

third and fourth experiment in 1L bottles. Survival was only measured in the small-scale test

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while the larvae in the bottle experiment were also used for tissue collection. Samples from

the second and third experiment were used to measure AMPs gene expression and PO

activity, respectively. The fourth experiment was carried out to evaluate a wider time

window of gene expression for the different AMPs (till 48 h, based on the results of second

experiment, in which the signal of expression was low).

Each treatment was performed in triplicate, each experiment in TCD test was repeated at

least once, and only one result is presented in this study. Plate sterility of the control

treatments was checked at the end of the challenge by plating 100 µl of the culture water on

LB35 plates and incubating at 17 °C. If the control was contaminated, the results were not

considered, and the experiment was repeated.

5.2.3.1. Experiment 1

A completely randomized experimental design was followed to evaluate the effect of PHB-A

on V. coralliilyticus exposure in mussel larvae. The challenge test was performed in 48-well

TCDs. Each well contained approximately 100 two-day-old D-larvae. The rearing water

contained LB35 (0.1% v/v) and rifampicin (10 mg L-1) to suppress growth of ambient bacteria.

In treatment 1 (control), larvae were neither PHB-A treated nor challenged, in treatment 2

the mussel larvae were challenged after 6 h with the rifampicin resistant strain

V.coralliilyticus at a concentration of 105 CFU mL-1 while in treatment 3 the larvae were

supplemented with PHB-A at a concentration of 1 mg L-1. PHB-A consists of a freeze-dried

Ralstonia eutropha culture containing 75% PHB on cell dry weight (VITO, Mol, Belgium). In

treatment 4, the larvae were supplemented with PHB-A 6 h before being challenged with

V.coralliilyticus. During the challenge test, larvae were pipetted twice a day to ensure that

PHB-A particles were suspended in the water column and available for uptake by the larvae.

The survival of the larvae was determined 24h and 48h after the addition of the pathogen.

5.2.3.2. Experiment 2, 3 and 4

The larvae were submitted to the same treatments as in Experiment 1, control PHB-A unfed

larvae were compared with PHB-A fed larvae challenged with 105 CFU mL-1 of V.

coralliilyticus. Three replicate glass bottles of 1L, containing 50,000 D-larvae each, were

sacrificed at each sampling point. Room temperature was 17°C. All the larvae of the three

replicate bottles were collected separately on a 60 µm sieve. For experiment 2, mussel

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larvae were pretreated with PHB-A for 6h before exposure to the pathogen and samples

were collected at the sampling points 3h, 6h, and 12h (counted just after adding the

pathogen), and no sampling was done at 24h and 48h. For experiment 3, larvae were

collected at 48h after the addition of the pathogen and PO was measured in challenged

larvae at 12h, 24h, 36h and 48h while for experiment 4, the larvae were collected at 0h, 3h,

6h, 9h, 18h, 24h and 48h. The larvae were washed with AFSW and transferred to 2 ml

centrifuge tubes, weighed, flash-frozen in liquid nitrogen and stored at -80°C for PO

measurement and RNA extraction respectively. Procedure of four experiment was

summarized in the Table below:

Table 5. 1: Summary of the challenge tests with V. coralliilyticus (105CFU mL-1) in the

presence of PHB-A (1 mg L-1) added 6 h before the pathogen

Challenge

experiment

(Exp.)

Vessel Sampling point Evaluation

Exp.1 Tissue culture

Dishes (TCD)

48h Survival of larvae

Exp.2 Glass bottle of 1 L 0, 3, 6, 9, 12, 24, 48, 72

and 96 h

Time window

Exp.3 Glass bottle of 1 L 0, 3, 6 and 12 h AMPs gene expression

Exp.4 Glass bottle of 1 L 12,24,26 and 48h Phenoloxidase activity

5.2.4. Standardization of quantitative real-time qRT-PCR for mussel larvae

5.2.4.1. Design of gene-specific primers

The sequences of mytimycin, mytilinB, defensin, and lysozyme from two blue mussel species

M. gallloprovincialis and M. edulis were selected from the GenBank

(http://www.ncbi.nlm.nih.gov). Specific primers for 18srRNA, mytimycin, mytilinB, lysozyme

and defensin genes were designed using the Primer3.0 software

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(http://biotools.umassmed.edu/bioapps/primer3). In this study, the 18srRNA gene was

retained as the housekeeping gene.

5.2.4.2. Gradient PCR

The stock and working solutions of the primers were made at concentrations of 100 µl and

20 µM respectively. The gradient PCR was performed with Bio-Rad My CyclerTM thermocycler

at temperatures varying between 52 and 66°C. Pure species of PCR products were obtained

by electrophoretic separation. The PCR products were run on a 2% agarose gel in 0.5X TBE

(Tris-borate-EDTA) buffer for 75 minutes at 400 volts.

5.2.4.3. Bulk PCR and Band elution of PCR product

To have sufficient amounts of PCR product for cloning and sequencing a total of 50 µl of the

reaction at each particular temperature was loaded and ran on a 1.5% agarose gel in 1X TBE

(Tris-borate-EDTA) buffer stained with Gel Red. The specific bands of interest were cut and

purified from gel slices, following the instructions of the manufacturer (Wizard SV Gel and

PCR Clean –up System, Promega, USA).

5.2.4.4. Cloning

Ten ligation mixes were prepared in advance, containing 5 µl ligation buffer, 1 µl vector, 3 µl

insert, and 1 µl T4 enzyme. 50 µl of thawed TOP10 competent E.coli was supplemented with

2 µl of a ligation mixture and incubated on ice for 20 minutes. Further, a heat shock of 42°C

was applied for 70 seconds after which the E.coli were immediately placed back in the ice.

950 µl of SOC medium (Tryptone 2%, Yeast Extract 0.5%, NaCl 0.05%, KCl 0.02%) was added

and the pH adjusted to at 7. Next, 1.8 ml of filtered glucose 20% and 0.5 ml filtered MgCl2

(2M) was added, and the mixture incubated at 37°C at 210 rpm for 1hour. Afterward, a

sample of 30, 150, 300 µl was spread on LB supplemented ampicillin (100 mg ml-1) plates

and grown in an incubator at 37°C for 16 hours. A single colony was picked up, and colony

PCR was performed to see only for positive clones. These clones were picked and grown

overnight in LB broth containing ampicillin.

5.2.4.5. Plasmid extraction and sequencing

The protocol for plasmid extraction is described by the manufacturer of the kit (Wizard Plus

Minipreps DNA purification system, Omega). NanoDrop 2000 (Thermo Scientific) measured

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the concentration of that plasmid. The number of plasmid copies was calculated according

to the formula of Whelan et al. (2003) and used to build the standard curve for each gene.

The concentration of the primers was between approximately 10 and 20 ƞg µl-1 and the final

volume was 10 µl. Primers T7 and SP6 were used as reverse and forward sequencing

primers, respectively. The genomic sequencing company LGC (Germany) conducted the

sequencing. The software Vector NTI version 15.0 was used to edit the sequence database.

The selected primer sequences were blasted on NCBI to verify their specificity for the

respective AMPs genes.

5.2.5. Real-time qPCR standardization of primer concentrations

The concentration of genes was optimized based on the manufacturer’s instructions

(Thermo Scientific Maxima SYBR Green/ROX qPCR Master Mix (2X)). Each primer was

prepared at different concentrations varying from 0.1 to 0.35 µM to select the optimal level

for defining the standard curve. A standard curve was drawn by plotting the natural log of

the threshold cycle (CT) against the natural of the number of molecules. The plasmids were

serially diluted from 102 to 106 plasmid copy µl-1. The standard curve was run in triplicate per

sample in the StepOneTM Real-Time PCR System thermal cycler (Applied Biosystems,

Belgium). The concentration of the primer that gave an optimal efficiency of 90 – 110 % with

a slope between -3.1 and -3.6 was selected to determine the standard curve for CT value

calculations.

5.2.6. Total RNA extraction and Reverse Transcription

Before the RNA extraction, the larvae were homogenized using the stomacher homogenizer

machine (Minibeabbeater, Biospec products, Branson 1200 Model B1200E-1, USA). Tissue

debris of the larvae was removed using the Qiashredder apparatus (Qiagen, Hilden,

Germany) to avoid clogging of the RNA extraction columns. Total RNA was extracted using

the SV Total RNA Isolation System kit (Promega, USA) following the manufacturer’s

instructions. Extracts were subsequently treated with DNase I (Fermentas, Germany) to

remove the remaining DNA. The RNA concentration was checked with the NanoDrop 2000

(Thermo Scientific) and adjusted to 500 ng µl-1 in all samples. The complete DNA degradation

within the RNA samples was confirmed by running the DNase-treated RNA sample in the

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PCR. After confirmation of the RNA quality by electrophoresis, it was stored at – 80oC for

subsequent use.

Reverse transcription was performed with a RevertAidTM H minus First strand cDNA

synthesis kit (Fermentas GmbH, Baden-Württemberg, Germany) following the

manufacturer’s instructions with some modifications (Ruwandeepika et al., 2011). Briefly, a

mixture of 1 μg RNA and 1 μl random hexamer primer solution was prepared. Then, 8 μl of

the reaction mixture containing 4 μl of 5x reaction buffer (0.25 mol−1 Tris-HCl pH 8.3, 0.25

mol−1 KCl, 0.02 mol−1 MgCl2, 0.05 mol −1 DTT), 2 μl of 0.01 mol−1 dNTP mix, 20 units of

ribonuclease inhibitor, 200 units of RevertAidTM H minus M-MuLV Reverse Transcriptase

was added. The reaction mixture was incubated for 5 min at 250C followed by 60 min at

420C. The reaction was terminated by heating at 700C for 5 min and then cooled to 40C.

cDNA samples were checked by PCR and stored at −200C for further use.

5.2.7. Real-time PCR

Real-time PCR was used to quantify the level of expression of the selected antimicrobial

peptide genes. This procedure was performed using Maxima® SYBR Green/ROX qPCR Master

Mix (Fermentas, Fisher Scientific, Erembodegem, Belgium) as described previously by Yang

et al. (2014) with some modifications. Briefly, the reaction was performed in a StepOneTM

Real-Time PCR System thermal cycler (Applied Biosystems, Belgium) in a total volume of 25

μl, containing 12.5 μl of 2 × SYBR Green master mix, 300 nM of forward and reverse primers

and 2 μl of template cDNA. The thermal cycle parameters used for the real-time

amplification were an initial activation at 500C for 2 minutes, initial denaturation at 950C for

10 min followed by 40 cycles of denaturation at 950C for 15 s and primer annealing at 600C

(gene 18srRNA), 630C (genes mytimycin, and mytilinB) , and 58oC (genes lysozyme and

defensin) and elongation at 60oC for 1 min. Dissociation curve analysis in the real-time PCR

was performed to check for the amplification of untargeted fragments. Data acquisition was

carried out with the StepOneTM Software.

5.2.8. Real-time PCR analysis (2-ΔΔCT Method)

The real-time PCR was validated by amplifying serial dilutions of cDNA synthesized from 0.5

μg of RNA isolated from mussel larvae samples. Serial dilutions of cDNA were amplified by

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real-time PCR using gene specific primers. ΔCT (average CT value of target – average CT value

of 18srRNA) was calculated for the different dilutions and plotted against the cDNA

concentration. The slope of the graph was almost equal to 0 for all four target genes.

Therefore, the amplification efficiency of reference and the target genes was considered to

be equal. Based on this precondition, real-time PCR data were analyzed using the 2-ΔΔCT

method (Schmittgen and Livak, 2008).

The relative expression was calculated as Relative expression = 2-ΔΔCT.

With ∆ΔCT = ∆CT,target – ∆CT, control

∆CT,target = CT, target, time x – CT, 18srRNA, time x

∆CT, control = CT, control, time 3 – CT, 18srRNA, time 3

RNA extracts from unchallenged mussels taken at 3h were used as a reference: expression in

this sample was set at 1 and all other data were normalized accordingly. The 18SrRNA gene

was used as a reference gene.

5.2.9.Phenoloxidase activity

The protocol as reported for Artemia Baruah et al. (2011) was slightly modified for use in

mussel larvae (De Rijcke et al., 2015). Briefly, based on sufficient cellular material (> 0.1 g),

the enzymatic transformation of L-3, 4-dihydroxyphenylalanine (L-DOPA) to dopachrome can

be used to determine the phenoloxidase (PO) activity of tissues. Before the actual PO

analysis, the homogenizing buffer (pH 7.5) was made (0.43% NaCl, 1.25 mM EDTA, 0.5%

Triton-X, 5 mM CaCl2). With this buffer, a 0.5 mM stock solution of L-DOPA was prepared (all

chemicals purchased from Sigma-Aldrich, Germany). Then, larvae (10% w:w) were

homogenized in the buffer using a pestle and stored overnight at 40C. Tube filters (0.22 µm)

removed cellular debris the following day through centrifuging (10,000 g, 20 min). Triplicate

20 µl aliquots of the resulting larval protein extracts were placed in a 96-well tissue culture

plate, and 200 µl of the L-DOPA solution was added to each well. Blank homogenizing buffer

samples were included for determining the non-enzymatic dopachrome production. The

tissue culture plates were dark-incubated at 300C, and absorbance (λ = 490 nm) was

measured twice a day for the next 48 hours using the Tecan spectrophotometer (Tecan I-

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Control). For all treatments, differences in average optical densities at time t and 0 were

calculated.

5.2.10. Statistics and analysis

Data analysis was carried out using the Statistical Package for the Social Sciences (SPSS

version 23). Statistical significance of differences in survival between treatment groups was

assessed using a one-way analysis of variance (ANOVA) followed by Tukey's post hoc test.

To study the effect of the treatments on expression levels (∆CT) of the different genes and on

PO a repeated measures ANOVA taking account of the replicate measurements was

performed, with treatment and time as factors. The significance of the interaction between

treatment and time was tested. In case the interaction effect was significant, a one-way

ANOVA or t-test was performed for each time point followed by post-hoc tests with

Bonferroni correction. For all statistical analysis a 5% significant level was used. The changes

in expression between a 0.5 and 2- fold threshold are often not considered as biologically

relevant (Figure 5.2, Figure 5.4, and Table 5.3).

5.3. Results

5.3.1. Survival

Survival of mussel larvae in Experiment 1 at 6h, 12h, and 24h was 100% for all treatments

(data not shown). However, significant differences were observed between the treatments

48h post-exposure, whereby the addition of PHB-A lowered the mortality for the larvae

challenged with V. coralliitycus (L+VC+PHB-A) compared to challenged larvae without PHB-A

treatment (L+VC). The survival was highest for non-challenged larvae L (CT) and L+PHB-A

(Figure 5.1).

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Figure 5. 1: Experiment 1. Survival (%) of mussel larvae after 48h exposure to V. coralliilyticus (105 CFU mL-1) in the presence or not of PHB-A (1 mg L-1). Data are expressed as mean ± Standard Error (SE) of three replicates. Different letters indicate significant differences (p < 0.05).

5.3.2. Design of immune gene primers for blue mussel larvae

Table 5.2 gives an overview of the primers that were designed to evaluate the gene

expression of the 4 selected antimicrobial peptides in mussel larvae by qPCR, using 18srRNA

as a housekeeping gene. A standard curve based on the amplification efficiency for each

gene was established (Table 5.3).

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Table 5. 2: Characteristics of the primers used for expression analysis with RT-qPCR

No Gene GenBank

accession no.

Primer Amplicon

length (bp)

Annealing

temperature

1 18srRNA L33448.1 F: TTAAGAGGGACTGACGGGGG 93 60 °C

R: TTGGCAAATGCTTTCGCAGT

2 Mytimicin JN825739.1 F: CCATTGTTGGGACTGCACTG 123 63 °C

R: CGGTCCCCACGTTCATAACA

3 MytilinB AF177540.1 F: CAGAGGCAAGTTGTGCTTCC 125 63 °C

R: GGAATGCTCACTGGAACAACG

4 Lysozyme AF334662.1 F: CCAACGACTATTCATGTGCCT 122 58 °C

R: TCCCCTTGGACCTCCATTGT

5 Defensin JN935271.1 F: CCCAGCACCGATTCTAGGAC 140 58 °C

R: AAGCGCCATATGCTGCTACT

Table 5. 3: RT-qPCR amplification efficiencies for all immune genes

No. Gene Funtion Slope R2 Amplification efficiency (%)

1 18SrRNA Housekeeping gene -3.5 0.99 93

2 Mytimycin Target gene -3.2 0.99 103

3 MytilinB Target gene -3.1 0.98 109

4 Lysozyme Target gene -3.3 0.98 100

5 Defensin Target gene -3.3 0.98 102

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5.3.3. Impact PHB-A and/or pathogen on the expression of the antimicrobial peptides

(Experiment 2).

The addition of the pathogen (L+VC) led to a significant down regulation of mytimycin after

12 h in comparison to the control treatment. Remarkably none of the genes was upregulated

upon Vibrio challenge neither at 6 nor 12 h. The addition of PHB-A on the other hand led to a

significant up-regulation of mytimycin and defensin after 6 h relative to the control. This

effect disappeared after 12h (Figure 5.2A & D). After 6 h, in the presence of Vibrio, PHB-A

down regulates mytimycin and lysozyme. After 12 h this is only the case of lysozyme. The

levels of defensin gene expression were equal for all treatments and time points except for

treatment L+PHB-A+VC at 6h post-exposure (Figure 5.2D). The overall expression pattern,

across treatments and time points, is rather similar for mytimycin and defensin. Both Vibrio

challenge and PHB-A seem to down regulate lysozyme expression at both time points.

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Figure 5. 2: Experiment 2. Expression of immune genes (A) mytimycin, (B) mytilinB (C) lysozyme, and (D) defensin in unchallenged (L) and challenged (L+VC) mussel larvae with V.coralliilyticus at 105 CFU mL-1 (n=3, mean ± standard error). PHB-A (1 mg L-1) was added to the mussel larvae culture water 6 h before the pathogen. The expression of AMP genes in the control treatment (CT) was regarded as 1.0. Different small and capital letters indicate significant differences between the treatments for the sampling points 6 h and 12 h post challenge respectively. Asterisks denote a significant difference between sampling point 6 h and 12 h post challenge for a particular treatment (T-test, P < 0.05).

5.3.4. Phenoloxidase activity (Experiment 3)

PHB-A significantly increased the phenoloxidase activity during the 48h of the assay (Figure

5.3). However, not only PHB-A but also V.coralliilyticus triggered a significant increase in

larval phenoloxidase activity (p < 0.05) as compared to the negative control (unchallenged).

Especially, PO activity was significantly increased in the double treatment where the

presence both of PHB-A and pathogen resulted in a higher PO activity relative to the control

but also to the single treatments.

Figure 5. 3: Phenoloxidase activity (PO) in the mussel larvae collected after 48 h exposure to the different treatments (mean ∆OD±SE, n=3). Data represent the increase in dopachrome over a 48 hours time frame in which PO activity in the 4 samples was measured. Data are expressed as mean ± Standard Error (SE) of three replicates. Different letters indicate significant differences for a given time point (P < 0.05).

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5.3.5. Time window of gene expression of AMPs (Experiment 4)

Overall, the expression of the AMPs genes in exposed mussel larvae appeared to be up-

regulated during the challenge test with V.coralliilyticus and to rapidly increase from 24h

onwards following exposure (Figure 5.4 A, B, and D). At 48 hrs, mytimycin, defensin, and

mytilinB are expressed 53, 35.7 and 8.3-fold higher respectively, in comparison to the

expression at time point 3h. During the 48h challenge test, the expression levels of the

lysozyme are down-regulated (Figure 5.4C) except at sampling point 6h, when an up-

regulation was noticed (Table 5.4). Differences between treatments because of a challenge

at subsequent sampling point are shown Table 5.3.

Figure 5. 4: Expression of immune genes (A) mytimicin, (B) mytilinB, (C) lysozyme and (D) defensin in challenged mussel larvae with V. coralliilyticus at 105 CFU mL-1 (n=3, mean ± standard error). The expression of AMPs gene in the negative control was regarded as 1.0 at 3h.

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Table 5. 4:The outcome of independent t-test between challenged and un-challenged mussel

larvae

Gene Post exposure (h)

6 9 12 18 24

Mytimycin *↑ *↑

MytilinB *↓ *↓ *↑

Lysozyme *↑ *↓ *↓

Defensin *↑ *↓ *↑

Asterisks denote a significant difference between challenged and un-challenged larvae at a particular sampling point relative to time point 3 h after exposure (t-test, p < 0.05). Symbols represent an up-regulation (↑), or down-regulation (↓). The sample of unchallenged treatment at 48h was not analyzed. The changes in expression between a 0.5 and 2- fold threshold are often not considered as biologically relevant, and are hence not included in the table. Difference in AMP expression in samples of challenged and unchallenged mussel larvae are verified by t-test at P < 0.05.

5.4. Discussion

The pathogenicity of this V. corallilyticus strain was again established in this study,

confirming the validity of the strain as a pathogen using the described challenge protocol.

Knowledge on the immune system in bivalve larvae is scare. In order to develop a tool box

for studying the immune response in bivalve mollusk, several studies concentrated on

designing specific primers for AMPs genes. Adults and larvae of M. galloprovincialis have

been studied quite extensively: primers for mytilin, myticin, defensin, heat shock protein

gene 70 (HSP70), mytilinB, lysozyme and for the house-keeping genes 18srRNA and 28srRNA

have been reported (Mitta et al., 1999a, Mitta et al., 1999b, Mitta et al., 2000a, Mitta et al.,

2000b, Cellura et al., 2007, Li et al., 2008).

The primers developed for M. galloprovincialis were tested at several occasions during this

research and did not work well for the blue mussel M. edulis larvae, challenged with V.

coralliilyticus. In addition, the time window of gene expression of AMPs in the larval phase of

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M. edulis is not determined yet. In fact, there is no information on the gene expression of

mytimycin in this species although it is abundantly present in the hemolymph of M. edulis.

Therefore it was necessary to first develop the primers for the 3 antimicrobial peptides that

were reported to be present in M. edulis adults (Charlet et al., 1996) as well as for the

hydrolytic enzyme lysozyme.

The selection of the housekeeping gene 18srRNA for mussel larvae appeared to be the right

choice, based on its expression in both untreated and treated mussel larvae and its stability

in expression, independently from the treatment (data not shown). This allowed us to

establish the kinetics of expression of the AMPs genes and lysozyme gene in blue mussel

larvae in response to a V.coralliilyticus challenge.

Phenoloxidase activity is commonly found in invertebrates (Smith and Söderhäll, 1991) and

is present in hemocytes of the adult mussel and larvae as well (Coles and Pipe, 1994,

Dyrynda et al., 1995). PO activity have been detected in all the disaggregated larval cells

from M. edulis, showing a stronger reaction in the veligers (Dyrynda et al., 1995) and also

previously observed in cells from the inner mantle fold of the pediveliger of O. edulis,

(Cranfield, 1974). Larval M. edulis cells are described to be capable of phagocytosis (Dyrynda

et al., 1995). Bacterial infection can cause a significant increase in PO activity in the whole

hemolymph of adult bivalves Crassostrea madrasensis and Chlamys farreri (Cong et al., 2008,

Gijo Ittoop et al., 2010). Recently De Rijcke et al. (2015) determined a significant increase in

PO activity in blue mussel larvae challenged with the toxin of harmful microalgae. The

implications of an elevated larval PO activity are, however, largely unknown as the

immunological role of PO is still poorly understood.

In this study, PO activity was detected in homogenates of M. edulis larvae and results show

that PO enzyme activity was elicited in non-infected mussel larvae in response to larvae fed

PHB-A. These findings are consistent with previous studies in other aquaculture species such

as Artemia (Baruah et al., 2011) and may at least partly explain why mussel larval survival

increased when PHB-A was added to the rearing water in this study (Figure 1), also observed

in previous experiments (Hung et al., 2015).

For a long time, mollusk hemocytes have been reported to be responsible for bactericidal

activities mediated against numerous toxic compounds (Li et al., 2008), such as

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antimicrobial peptides (Charlet et al., 1996) and lysozyme (McHenery et al., 1979), and by

phenoloxidase activity (Coles and Pipe, 1994). During the last decade, knowledge of immune

processes in adult bivalves have been significantly improved by the development of genomic

tools (Fleury et al., 2009, De Lorgeril et al., 2011, Fleury and Huvet, 2012). However, the

immune characteristics of larvae remain under-investigated, notably due to the difficulty to

isolate hemocytes from larvae. Elston (1980) observed phagocytes (described as

coelomocytes) containing bacterial fragments in the visceral cavity of C. virginica veliger

larvae. Dyrynda et al. (1995) have confirmed that some elements of the immune system in

adult M. edulis are also present in the trochophore and veliger larvae of this species.

Recently, several studies reported that hemocytes appear during the gastrular –

trochophore developmental stages. At these stages, haemocyte generation/proliferation

and induction of immune related genes are concomitant (Tirapé et al., 2007). In the mussel

Mytilus galloprovincialis, the antimicrobial peptides mytilin and defensin have been found

during and after larval metamorphosis (Mitta et al., 2000a). Furthermore, lysozyme-like and

hydrolysis enzyme activities are present in C. gigas larvae. Bivalve hemocytes seem to

respond to bacteria stimulation with a burst of respiratory activity similarly to the

respiratory burst of mammalian phagocytes, resulting in the generation of various free

radicals or reactive oxygen species (ROS) that eliminate the phagocytized material

(Labreuche et al., 2006, Lambert et al., 2007). ROS production are controlled by antioxidant

defense systems to limit tissue peroxidation (Genard et al., 2013). The generation of ROS

metabolites has been recorded in both trochophore and D-larvae cells of M. edulis (Dyrynda

et al., 1995).

Genard et al. (2013) reported that bacterial infections can have serious consequences for the

survival of bivalve larvae. When a pathogen infects a host, multiple reactions occur, initiated

both by the pathogen in an attempt to survive and multiply, and by the host in an attempt to

eliminate the pathogen. For the host, changes induced by infection can be seen at several

levels such as molecular, physiological and biochemical processes. First, infection induces

the activation of both cellular and humoral immune responses that act together to kill and

eliminate the infecting bacteria. In bivalves, immunity is constituted of innate processes

including various serologically active molecules including antimicrobial factors and of the

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phagocytosis accompanied by production of oxygen metabolites and the release of

lysosomal enzymes.

After engulfment of microorganism, the phagosome undergoes maturation with acidification

and sequential fusion with endosomal and lysosomal compartments including granules,

which contain diverse families of antimicrobial peptides/proteins (Gueguen et al., 2009,

Schmitt et al., 2012). The release into the phagosome of microcidal compounds leads to the

rapid neutralization/degradation of the engulfed microorganisms. Among the hydrolytic

enzymes that are released into the maturing phagosome, lysozymes are known play an

important role in microbial destruction due to their lytic properties on the peptidoglycan of

the bacteria cell wall (Hancock and Scott, 2000). It is likely that AMPs and lysozymes stored

in hemocyte cytoplasmic granules are delivered to the phagosome to kill phagocytosed

bacteria (Bachère et al., 2015).

The existence and diversity of AMP has been revealed both in M. edulis (Charlet et al., 1996)

and M. galloprovincialis (Hubert et al., 1996). Cellura et al. (2007) described how AMPs

respond specifically to the challenges, confirming that at least some of the innate immune

mechanisms are specifically orientated. Mytimycin is linked to fungal infection (Charlet et al.,

1996, Sonthi et al., 2011) and also shows very low expression in the velum of M.

galloprovincialis larvae, when challenged with Vibrio anguillarum (Balseiro et al., 2013). This

is in contradiction to the earlier findings of Mitta et al. (2000a) who found that both mytilinB

and defensin genes in M. galloprovincialis are constitutively expressed and not inducible

following Vibrio alginolyticus (DSMZ 2171) bacterial challenge (possibly the host response is

strain dependent). In addition, the expression of both genes mytilinB and defensin are

recorded to be developmentally regulated, and neither gene is expressed in mussels until

after larval settlement and metamorphosis according to Mitta et al. (2000a). This study,

therefore, focused on immune gene expression in D-larvae both by PHB-A and Vibrio

coralliilyticus at two specific time points namely 6 and 12 h after challenge. Overall the

results seem to indicate that in general in this short time frame, neither PHB-A nor challenge

seem to upregulate the measured immune genes (apart from some upregulation after 6h by

PHB-A) This might indicate that the infection process had not started yet (in which case the

fluctuations are stochastic) or that this particular species is able to control the host response

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Chapter 5 PHB-A regulates immune response in challenged mussel larvae

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at the transcriptional level (assuming that infection had started). The PHB-A treatment did

not have a major positive effect either and hence does not seem to contribute to the

capacity of the host to maintain expression of the tested genes (in this time frame). In view

of these observations immune genes expression was monitored over a longer time frame.

An experiment running over 48h demonstrated that mussel larvae (2-day old D-larvae)

regulate the tested genes from 24h post-exposure onwards. Mytimycin is the AMPs gene

with the highest expression in M. edulis larvae in response to the exposure to the pathogenic

bacterium V. coralliilyticus, followed by defensin and mytilinB. This result indicates that D-

larvae is either responding very late to the invading pathogen or that the pathogen is only

invading the host after 24 h exposure when an increased expression of the tested genes

becomes apparent. This is also in agreement with the results of the first experiment of this

study where mortality only occurs after 48 h. The former explanation would be in

accordance to the report of Balseiro et al. (2013) who stated that bivalve larvae are not

entirely immune-competent to combat infections by pathogens.

However the PO activity measurement, in larvae 48 hours after exposure, seems to point in

the other direction. Here PHB-A stimulates PO, possibly contributing to the capacity of the

larvae to handle a Vibrio challenge, in which case PHB-A acts as an immunostimulant. PO

activity is also increased upon Vibrio exposure, but that level of PO activity is insufficient for

protection against Vibrio. A double exposure, PHB-A, and Vibrio, increased PO activity even

further. These data seem to suggest that some immunological capacity is available in this

developmental stages and that it can be steered, such as by the addition of PHB-A.

Taken together it is apparent that the immunological response in larvae as monitored by

gene expression can only be interpreted at its best if simultaneously the infection process is

visualized (by e.g. immunohistochemistry). Such data would allow a better understanding of

the dynamics of the immune response.

In conclusion, this study reports the successful development of 5 primers (3 AMPs, 1

hydrolytic enzyme, 1 house-keeping gene) to monitor the immune response of blue mussel

(M. edulis) larvae. PHB-A regulated immune gene expression was found in challenged mussel

larvae, as revealed by PO activity in unchallenged and challenged (48h after exposure)

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Chapter 5 PHB-A regulates immune response in challenged mussel larvae

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larvae. This may at least partly explain why larval survival increases in the presence of PHB-

A.

It was demonstrated that 2 day-old blue mussel larvae activate the expression of the three

AMPs, mytimycin, mytilinB and defensin upon invasion by the pathogen V. coralliilyticus. The

level of expression is up- and down-regulated in the first 12hours after exposure in an

unclear pattern, but rapidly increases from 24 to 48 h post-exposure. The expression of

lysozyme, however, remains very low and stable during the first 48 h after challenge.

Mytimycin gene is one of the genes with the strongest expression in blue mussel larvae.

Acknowledgments

The Vietnam International Education Development (VIED) and Vlir-OI project (“Ensuring

bivalve seed supply in Central of Vietnam”, 2011-068) financial supported this work through

a doctoral grant to Nguyen Van Hung. The Research Foundation - Flanders (FWO, Belgium),

supports Peter DS as a post-doctoral research fellow. The guidance and assistance of the

staff of the Artemia Reference Center (Ghent University, Belgium) are highly appreciated.

We wish to thank Dr. Linsey Garcia-Gonzalez (VITO, Belgium) for providing the PHB-A. Dr.

Kesarcodi-Watson (Cawthron Institue, New Zealand) kindly provided the bacterial strain V.

corallilyticus. We wish to thank Dr. Abatih Emmanuel (FIRE, University of Ghent) for

statistical guidance.

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GENERAL DISCUSSION

CHAPTER 6: GENERAL DISCUSSION

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Chapter 6 General discussion

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6.1. Introduction

In bivalve aquaculture species, susceptibility to viral and bacterial infections is higher in

larvae than adults (Lane and Birkbeck, 2000, Arzul et al., 2001). Major knowledge gaps still

exist in our understanding of the internal defense mechanisms in the early life-history of

bivalves. To date, most immunological studies in bivalves have been performed on adults,

probably because it is not possible to obtain blood samples from larvae and young juveniles

needed to study the hemocytes responsible for the internal defense (Luna-González et al.,

2003). Several studies reported though that hemocyte appears during the gastrular –

trochophore developmental stages and that, hemocyte generation/proliferation and

induction of immune related genes are concomitant at these stages (Tirapé et al., 2007).

Therefore, this study accepted the challenge and concentrated on the gene expression of

immune defense effectors in blue mussel (Mytilus edulis) larvae. Advances in

characterization of these effectors in larvae may lead to a better understanding of the

immune defense mechanisms and give new insights into health management and diseases

control in the larval culture of bivalves.

Polyhydroxybutyrate (PHB) is a typical storage compound of carbon and energy that occurs

widely in prokaryotes. This polymer can be accumulated up to 90% of the cellular dry weight

of some bacteria (Uchino et al., 2008). PHB can be degraded extracellularly by many types of

bacteria that secrete specific extracellular PHB depolymerases into the environment or by

intracellular mobilization of PHB in the accumulating strain itself. Originally, the interest for

PHB was based on its medical and industrial application possibilities (Anderson and Dawes,

1990, Chen and Wu, 2005), and it was not until recently that PHB was identified as a

potential bio-control agent for aquaculture (Defoirdt et al., 2007b). The use of crystalline

PHB (i.e. extracted from the bacterial cells, PHB-C) as a bio-control agent has been tested

and was found promising to control vibriosis in different aquaculture species (Nhan et al.,

2010, Sui et al., 2012). However, the efficiency of PHB was shown to be considerably higher

when supplied in the amorphous state (i.e. still contained in bacteria cell, PHB-A) (Halet et

al., 2007). Indeed, the impact of both forms of PHB on aquatic species is different due to a

number of confounding factors such as the particular shape, size, digestibility, and others (cf

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infra). In this study, PHB was applied as a pre-biotic compound in the larval culture of a

model bivalve species, the blue mussel Mytilus edulis (Figure 6.1). The effects of crystalline

and amorphous PHB were evaluated (1) based on the growth performance and survival of

mussel larvae and early-set spat under normal rearing conditions, (2) based on the

interference with virulence factors of mussel pathogens in vitro and the results of larval

challenge tests in vivo, and (3) based on the larval innate immune response. In this chapter,

our results are confronted with the existing literature and discussed in the light of research

needs and application opportunities in hatcheries.

Figure 6. 1: Schematic overview of the study on the mode of action of PHB in mussel larvae

culture.

6.2. The effect of PHB on bivalve larvae and spat performance

The successful application of PHB in aquaculture systems has been explored in different

species and at different life stages. Based on these results, it can be stated that the

beneficial effects of PHB, including acting as an antimicrobial agent and changing the

microbial composition in the digestive tract of the cultured animal, depend on several

Mussel larvae

PHB

Antimicrobal

activity/protection

Innate immune

response

Survival & growth

performance

Conclusion

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Chapter 6 General discussion

135

factors such as application method, a form of PHB, concentration and aquaculture species.

Depending on the feeding behaviour of the target species, PHB-C can be administered either

directly to the culture water (giant freshwater prawn larvae (Nhan et al., 2010)), as a

compound of formulated feeds (European seabass (De Schryver et al., 2010), Siberian

sturgeon (Najdegerami et al., 2012)) or as enrichment of live feed (giant freshwater prawn

larvae (Thai et al., 2014), Chinese mitten crab (Sui et al., 2014). Blue mussel larvae are active

filter feeders, so the direct application of both forms of PHB in the culture water proved to

be successful (Chapter 3).

The supplementation of PHB-A into the rearing water of blue mussel larvae led to better

performances than the supplementation of PHB-C. Indeed, this result may be due to their

inability to efficiently metabolize PHB-C (Chapter 3), despite their proven ability to ingest it.

In contrast, the addition of PHB-A at a concentration of 1.0 mg L−1 did have a positive effect

on the larval survival. This may be attributed to the fact that PHB-A is better biodegradable

and has a smaller size than PHB-C (Halet et al., 2007). At this stage the exact mechanism by

which the PHB polymer is broken down inside the intestinal tract of animals is not known,

i.e. whether it is mainly driven by physicochemical processes or by biological activity of the

host and/or microorganisms present in the gut (Nhan et al., 2010). Defoirdt et al. (2007b)

and Liu et al. (2010) suggested that the metabolism of PHB in brine shrimp nauplii occurs at

least partially by the enzymatic activity of the host. Based on the results of our study, it can

be hypothesized that PHB-degrading enzymes that would enable mussel larvae to digest the

compound are lacking since D-stage mussel larvae have an immature digestive system.

Supplementing the strongest PHB-degrading bacterium ARC4B5, isolated from PHB-treated

mussel larvae, to the larval cultures that received PHB-A, did not result in an increase in

survival, on the contrary, seemed to lower the growth performance of the larvae. This is in

contrast to the findings of Liu et al. (2010) who reported that the application of PHB-

degraders as pre/probiotic in combination with PHB was better than PHB alone. As a

consequence, it would seem that enzymes play a more important role in PHB-digestion than

microbial activity. However, it is not clear whether the immature digestive system of D-stage

larvae possess depolymerases.

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Chapter 6 General discussion

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It should also be kept in mind that PHB-A does not contain solely PHB, as is the case of PHB-

C, but also dead bacterial cell walls. The latter may also trigger an increased immune

response (Nathalie, 2001) and ultimately lead to less mortality. More and more evidence is

being obtained in other species for this alternative explanation of the observed beneficial

effect, basically indicating that PHB effects are multifactorial

The supplementation of PHB to the culture water of the mussel larvae did not result in faster

growth nor in an improvement of spat performance in terms of growth or survival regardless

form and concentration. In contrast, De Schryver et al. (2010) found that supplementing the

diets of European sea bass with 2% or 5% crystalline PHB led to a significant increase in fish

weight compared to fish fed the control feed (0% PHB). Nhan et al. (2010) and Thai et al.

(2014) demonstrated the faster development of giant freshwater prawn larvae fed with

Artemia nauplii enriched with crystalline or amorphous PHB respectively. Similarly, Sui et al.

(2012) showed a growth- promoting effect of crystalline PHB on Chinese mitten crab larvae.

The positive effect of PHB on fish growth can be explained by the fact that the monomer β-

hydroxybutyrate (β-HB) resulting from degradation (enzymatic or microbial) is known to be

used as carbon and energy source for fish (Kato et al., 1992, Patnaik, 2005, De Schryver et

al., 2010). Mussel larvae, however, do not seem to use PHB and/or its degradation products

as fuel for development and growth. I hypothesize therefore that either the mussel larvae

are not capable of degrading PHB or that the monomer β-HB is not used as a ketone body by

this marine bivalve in contrast to vertebrates and crustaceans (Weltzien et al., 1999). The

latter is supported by the findings of Stuart and Ballantyne (1996) who did not detect any

activity of the enzyme β-hydroxybutyrate dehydrogenase in the gills or digestive gland of

adult Mytilus edulis, which indicates that the mussels are not capable of producing nor using

β-HB. Besides, the lack of this enzyme seems to be general in marine mollusk. In other

words, whilst PHB seems to stimulate the immune system of blue mussel larvae resulting in

a positive effect on the survival this phenomenon has an energetic cost which affects

growth, and the production of ketones does not seem to compensate for that since marine

bivalves mollusks are not able to use β-HB ketones. All this requires further verification.

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Chapter 6 General discussion

137

6.3. The effect of poly-β-hydroxybutyrate on the microbial composition in the digestive

tract of blue mussel larvae

Establishing and maintaining beneficial interactions between the host and its associated

microbiota are critical requirements for host health. Although the gut microbiota has

previously been studied in the context of inflammatory diseases, it has recently become

clear that this microbial community has a beneficial role during normal homeostasis,

modulating the host’s immune system (Sommer and Backhed, 2013). Concerted research

efforts have concentrated on optimizing livestock production (both terrestrial and aquatic)

with eco-friendly alternatives to the therapeutic use of antimicrobials. In several previous

studies, PHB was used as a prebiotic in aquaculture species, offering some benefits to the

host primarily via the direct or indirect modulation of the gut microbiota. PHB interacts with

the microbiota inhabiting the gut and induces modifications at the microbial community

level and consequently affects the host level. In fact, the presence of PHB in the gut of the

animal has been demonstrated to induce a steering effect on the gut microbial community in

juvenile European sea bass (De Schryver et al., 2010), and in Siberian sturgeon fingerlings

(Najdegerami et al., 2012) where the gut pH decreased from 7.7 to 7.2 suggesting that the

presence of PHB in the gut led to the increased production of short-chain fatty acids (De

Schryver et al., 2010). Nhan et al. (2010) reported that supplementation of PHB in the

culture water resulted in a decreased number of Vibrio spp. in the intestinal tract of

freshwater prawn larvae.

In our research (Chapter 3), it was shown for the first time that PHB tends to alter the gut

microbiota in mussel larvae regarding relative abundance, dynamics, and genetic diversity.

Bacteria that are enhanced by PHB may represent niche populations able to metabolize PHB

during the gastrointestinal passage. The capacity to produce extracellular PHB depolymerase

is a widespread phenomenon amongst bacteria and was demonstrated under aerobic,

anaerobic and thermophilic conditions (Tokiwa and Ugwu, 2007). A total of 22 strains of PHB

degraders were isolated from the digestive system of mussel larvae and their degrading

properties demonstrated. Of these 22 strains, 16 PHB-degrading isolates seemed to belong

to the genus Pseudoalteromonas, of which isolate ARC4B5 showed the strongest PHB-

degrading activity.

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Chapter 6 General discussion

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Our study showed that the microbial community of mussel larvae fed PHB tended to be

more diverse than the control treatment, although not significantly. As a consequence, no

close link could be defined between survival and growth performance on the one hand and

the intestinal microbiota community structure, richness and diversity in the mussel larvae on

the other hand. This is in contrast to the findings of De Schryver et al. (2010) and

Najdegerami et al. (2012) who observed significant differences. A possible explanation

suggests that PHB degradation does not occur in the immature digestive tract of mussel

larvae because the transit time of the food particles is too short (Bayne et al., 1987). Mussel

larvae clear their gut in less than 2 hours which may prevent degradation of PHB both

enzymatically and microbially.

6.4. Effect of poly-β-hydroxybutyrate on disease resistance of bivalve larvae

Poly-β-hydroxyalkanoate polymers can be degraded into β-hydroxy short-chain fatty acids of

which β-hydroxybutyrate (β-HB) exhibits some antimicrobial, insecticidal, and antiviral

activities. The antimicrobial activity of β-HB is thought to be comparable to that of other

short chain fatty acids although the antibacterial mechanism(s) of these compounds are not

completely understood (Ricke, 2003, Tokiwa and Ugwu, 2007). The antimicrobial effect of β-

HB towards aquaculture pathogens Vibrio campbellii and Vibrio harveyi has previously been

investigated by Defoirdt et al. (2007b), who demonstrated that the growth-inhibitory effect

of β-HB was sensitive to the environmental pH. At pH 5, growth was completely inhibited, at

pH 6, growth was delayed and at pH 7, no inhibition could be observed (Defoirdt et al.,

2006b). It is probable that this pH-dependency can be explained by the fact that fatty acids

can pass the cell membrane only in their undissociated form, which is more prominent at

lower pH (Sun et al., 1998). In this study (Chapter 4), the effect of β-HB on the growth and

virulence of the bivalve pathogens was assessed in in vitro experiments at pH 7 and 8. It was

observed that β-HB affected the virulence factors’ activity of the bivalve pathogens rather

than the growth. The effect of β-HB on the virulence factors depended on the pathogenic

strain, the environmental pH and did not allow for a clear pattern to be distinguished. The

highest β-HB concentration (125 µM) inhibited some virulence factors of the pathogenic

bacteria such as hemolysis, phospholipase, but also stimulated caseinase in V. splendidus at

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Chapter 6 General discussion

139

pH7 and V. coralliilyticus at pH8. In addition, β-HB had no effect on gelatinase, lipase, and

biofilm formation. The concentration of 125 µM is biologically speaking irrelevant for mussel

larvae and hence the results should be put in the right perspective.

The effect of PHB-A on larval survival during challenge experiments depended on various

factors: the pathogen, PHB-A concentration and exposure time. In general, survival of mussel

larvae that received PHB-A at a concentration of 1 mg L-1 PHB-A 6h before the challenge test

was higher compared to the survival of the larvae that received the PHB-A pre-treatment

24h before the challenge test. Larvae that received PHB-A 6h before the challenge showed a

55% and 25% increase in survival measured 96h after exposure to the pathogens V.

splendidus and V. coralliilyticus respectively, as compared to the controls. Increasing the

pretreatment from 1 to 10 mg PHB-A L-1 did not further improve the results.

These results validate V. coralliilyticus and V. splendidus as being pathogenic for M. edulis

larvae. Furthermore, it was demonstrated that PHB-A as a pre-treatment (6h) offers

protection to the mussel larvae and increases survival significantly during challenge tests.

6.5. Antimicrobial peptides (AMPs) immune response in bivalve mollusks

Bivalves lack an adaptive immune system and, therefore, do not possess immune memory.

Alternatively, they have developed an innate immune system involving cell-mediated and

humoral components used to recognize and eliminate pathogens (Gestal et al., 2007).

Antimicrobial peptides (AMPs) are essential elements of the innate host response to

microbial invasion (Gestal et al., 2007). The study of antimicrobial peptides is important to

increase our basic knowledge on shellfish immunity, but also to evaluate the potential of

these peptides as disease management tools in aquaculture.

They are usually characterized by their small size, heat-stability and a broad range of

antimicrobial activity (Licheng et al., 2009). Antibacterial activity was first reported in

mollusks in the ‘80s (Kubota et al., 1985) whereas the isolation and characterization of true

AMPs from the mussels Mytilus galloprovincialis (Hubert et al., 1996) and M. edulis (Charlet

et al., 1996) date back to 1996. Taking into account the features of their primary structure

and their consensus cysteine array, these peptides were classified into four groups: i)

mytilins, with five isoforms (A, B, C, D and G1) (Mitta et al., 2000a, Mitta et al., 2000c), ii)

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Chapter 6 General discussion

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myticins, with three isoforms A, B (Mitta et al., 1999a, Mitta et al., 1999b), and C (Pallavicini

et al., 2008) iii) defensins, found in both mussel and oysters, with two isoforms (MGD1 and

MGD2) in M. galloprovincialis; defensin A and B in M. edulis, two isoforms in the Pacific

oyster, Crassostrea gigas (Cg-Def1 and Cg-Def2), and AOD (American oyster defensin) in C.

virginica (Charlet et al., 1996, Mitta et al., 2000a, Seo et al., 2005, Gonzalez et al., 2007)

and iv) mytimicin, partially characterized from M. edulis plasma (Charlet et al., 1996).

In a first research line (Chapter 5), three genes coding for the AMPs mytimycin, mytilin B,

defensin and one gene coding for the hydrolytic enzyme (lysozyme) were selected to study

the immune gene expression in blue mussel larvae when exposed to the pathogen V.

coralliilyticus. The mytimicin gene is one of the AMPs only present in M. edulis, but not in M.

galloprovincialis whereas mytilinB, and defensin exists in both Mytilus species (Tincu and

Taylor, 2004). A first challenge was to develop the primers for AMPs that work specifically

for blue mussel (M. edulis) larvae.

Next, these home-designed primers were used to optimize the Real-time PCR protocol for

blue mussel larvae and to investigate the immune gene response of the mussel larvae when

challenged with V. coralliilyticus, as opposed to the traditional approach where functional

assays and histological studies determine the lesions and the interactions between host

immune system and pathogens (Figueras and Novoa, 2004). To date, most of the

information on mussel larval immunity is based on phagocytosis assays (García-García et al.,

2008) chemo-illuminescence production and nitric oxide release (Costa et al., 2009).

Information on gene expression of the immune response in mussel larvae tissue

homogenates is scarce. Mitta et al. (2000a) analyzed the expression of the genes encoding

for the AMPs mytimicin, mytilin B, defensin, and lysozyme in the hemocytes of adult animals

subjected to various stress factors, as well as in tissue homogenates during larval

development.

Overall the results seem to indicate that in a short time frame (up to 12 h), neither PHB-A

nor challenge seem to up-regulate the measured immune genes (apart from some up-

regulation after 6h by PHB-A). This might indicate that the infection process had not started

yet (in which case the fluctuations are stochastic) or that this particular pathogenic strain is

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able to control the host response at the transcriptional level (assuming that infection had

started). The PHB-A treatment did not have a major positive affect either and hence does

not seem to contribute to the capacity of the host to maintain expression of the tested

genes (in this time frame). In view of these observations immune genes expression was

monitored over a longer time frame.

When considering a time frame of 48 h, the response of exposed mussel larvae appeared

soon after 3 hours, but a drastic increase in expression of mytimycin 48 h after exposure to

the pathogen as well as induced up-regulation of defensin and mytilinB from 24h post-

exposure onwards was observed. The latter is interesting since the expression of both genes

mytilinB and defensin are reported to be developmentally regulated, and not being

expressed in mussels until after larval settlement and metamorphosis (Mitta et al., 2000a).

These results are in agreement with the results of Gestal et al. (2007) who found that the

expression levels in carpet shells (Ruditapes decussates) increased from 24 to 48h after

exposure to a mixture of both dead and alive Vibrio anguillarum. When comparing the gene

expression of the 3 AMPs in infected mussel larvae, mytimicin was found to be highly

expressed, suggesting that the production of this AMP is quite important as a protection

mechanism in mussel larvae against invasive pathogenic bacteria.

This result indicates that D-larvae are either responding very late to the invading pathogen

or that the pathogen is only invading the host after 24 h exposure when an increased

expression of the tested genes becomes apparent. This is also in agreement with the results

of the first experiment where mortality only occurs after 48 h. The main clinical signs

observed in challenged scallop larvae for 24 h were bacterial swarms on the margins of the

larvae, extension and disruption of the velum, detachment of velum cilia cells and digestive

tissue necrosis (Beaz‐Hidalgo et al., 2010) and very high mortality (98 -100%) in challenged

oyster larvae less than 48 h (Rojas et al., 2015).

In addition, AMPs are found to be abundant in hemocytes which are an important barrier

defense in the mussel. Conversely, during pathogen exposure, the expression of the

lysozyme gene was stable and seemed not to be induced when the mussel larvae were

exposed to the pathogen V. coralliilyticus. This seems logical since lysozyme can be in both

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142

the gut and the digestive system of the mussel. However, lysozyme were able to

differentiate amongst bacteria species, even between two vibrio species. The expression of

lysozyme was downregulate to V. coralliilyticus whereas may be strongly modulate to

another stressors (Li et al., 2010). Further studies must address the behavior of the other

known immune molecules in order to obtain a complete overview of the innate response.

The main goal of the present research was to study the gene immune reactions produced

after stimulation with pathogenic Vibrio bacteria and PHB-A. This new information will

contribute to increased knowledge about the defense mechanisms in this species, which is

of major interest to the aquaculture sector.

6.6. Induction of phenoloxidase enzyme activity in the early life stage of bivalve larvae by

PHB.

Phenoloxidase (PO) activity is considered a constituent of the immune system and is

probably responsible, at least in part, for the defense mechanism upon non-self recognition

in crustaceans, insects (Hernández-López et al., 1996) and bivalve mollusks (Coles and Pipe,

1994, Dyrynda et al., 1995). PO is present in hemocytes of the adult mussel and also

detected in disaggregated larval cells from M. edulis, showing a strong reaction in the

veligers (Dyrynda et al., 1995). PO activities always had a strong relationship with pathogen

levels: f.ex. bacterial infection caused a significant increase in PO activity in the whole

hemolymph of the bivalves Crassostrea madrasensis and Chlamys farreri (Cong et al., 2008,

Gijo Ittoop et al., 2010). Recently De Rijcke et al. (2015) recorded a significant increase in PO

activity in challenged blue mussel larvae with toxic harmful microalgae.

The presence of PO in the hemolymph of bivalve mollusks, and its stimulation by bacterial

and fungal cell wall components in the adult mussel Perna viridis confirm that PO plays a role

in the internal defense mechanism (Deaton et al., 1999). The implications of an elevated

larval PO activity are, however, largely unknown as the immunological role of PO is still

poorly understood. The current study (Chapter 5) indicated that the addition of PHB-A

increased PO enzyme activity in the early life stages of blue mussel larvae. The combination

of V. coralliilyticus and PHB-A stimulated the PO activity in mussel larvae, indicating that

PHB-A treatment might facilitate a PO response to a pathogen.

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6.7. The proposed model for future application of amorphous PHB in bivalve larvae

culture.

Recently, under lab conditions, PHB has been successfully applied to some aquaculture

species, resulting in an increase in survival, grow performance and disease resistance. In this

thesis work, we demonstrated that the use of amorphous PHB-A was more advantageous

than crystalline PHB, leading to higher larval survival both under challenged and

unchallenged conditions. Possibly, the addition of the bacterial cell walls plays a role as well

as an immune-stimulating agent. This research indicated that the application of PHB as a

biological control against bacterial infection in bivalve mollusk aquaculture would be a valid

option, and certainly has potential benefits for the bivalve hatchery industry. It was

demonstrated that PHB-A acts mainly as an immune-stimulating compound, enabling the

blue mussel larvae to protect themselves at least partly against the invasion of pathogens.

The concentrations of PHB used in this study were significantly lower than those used for

other aquaculture organisms which make it even more attractive as a disease control tool.

The fact that PHB-A seems to have a positive role also opened the possibility to apply

microbial biomass rich in PHB rather than PHB-C. PHB-C needs to be chemically extracted

from PHB-rich microbial biomass, increasing the price of its production. A thorough

validation of the benefits of PHB-A at a commercial hatchery level might provide the

necessary data to verify the economic feasibility of PHB-A application.

6.8. General discussions

The survival of the mussel larvae almost doubled following the application of 1 mg L-1 PHB-A

to the culture water of blue mussel larvae. This research demonstrated that amorphous PHB

was more effective than crystalline PHB in mussel larvae culture. Both forms of PHB did not

improve larval growth or metamorphosis and appeared not to be used as an energy source

in mussel larvae. PHB-A can partly protect mussel larvae against the specific pathogenic

bacteria V. splendidus and V. coralliilyticus, but the effect depended on both PHB-A

concentration and exposure time. The beneficial effect of PHB-A could also be attributed to

the presence of the dead bacterial cell walls, but this needs further investigation.

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In general, pretreatment of mussel larvae with PHB-A at a concentration of 1 mg L-1 resulted

in better larval survival when pretreated 6h before the challenge test compared to being

treated 24h before the challenge test. Β-HB had some impact on selected virulence factors

of the pathogenic bacteria but there was no clear pattern, and the effects were only

observed at concentrations that were not biologically relevant. Interestingly, the

supplementation of PHB-A regulates gene expression in challenged mussel larvae but also

triggers increased PO activity. The AMP gene expression in mussel larvae challenged with V.

coralliilyticus was strongest from 24 h after challenge onwards. The expression of defensin is

for the first time described in blue mussel larvae. Based on these results we can make a

strong assumption that PHB-A has an effect on the immune system of the blue mussel larvae

rather than having an anti-bacterial activity against the pathogens.

6.9. Further perspectives

1. By linking the changes in AMP gen-expression with the actual production of the AMP

through Western blot (for which antibodies would be needed), one would gain a better

insight in the biological meaning of the registered up- and downregulation of the responsible

genes.

2. The expression of the AMP-genes has been described to depend on the nature of the

stressor. It would be very interesting to monitor gene expression and the effect of PHB when

comparing different stressors such as temperature shock and physical stress caused by

handlings common in hatcheries (sieving f.ex.). It would give a better insight in the diversity

of responses of the innate immune system of the mussel.

3. The regulation of AMP expression is species dependent. It would be interesting to run the

experiments of Chapter 5 with Japanese oyster larvae Crassostrea gigas, the most important

bivalve in terms of aquaculture production or with bivalve species that are renowned for

their stress-sensitivity such as scallops. The genome of C. gigas is almost completely known

and extensive literature is available on the genetic regulation of its innate immune system.

As a result, changes in AMP production caused by PHB could be linked to the responsible

genes.

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4. The importance of the incubation time with PHB before exposure to the pathogen was

obvious in our study. Best results were obtained with an incubation period of 6 h. Therefore

application of PHB-A as a pre-treatment at different time slots (e.g. 18 h) could be

interesting and should be related to a better understanding of the infection model of the

pathogen under investigation. Mode of infection can be followed visually and histologically

by fixating mussel larvae in parafilm at different time intervals. By labelling the pathogen

with a green fluorescent protein (f.ex. (GFP-HI-610) (Rekecki et al., 2012), one can localize

the pathogen in function of time (eventually immunohistochemistry techniques could be

used as well, provided antibodies could be raised against the pathogens. Tissue damage

could be visualized with standard staining methods (hematoxylin and eosin).

5. PHB-A contains not only PHB but also the bacterial cell wall. In addition, Ralstonia

eutropha is a gram-negative bacterium possibly containing endotoxins in the cell wall which

may also have an effect on mussel larvae when PHB-A is supplemented. Lee et al. (1999)

produced poly (3-hydroxybutyrate) by cultivating several gram-negative bacteria, including

Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. When PHB was

recovered by the chloroform extraction method, the endotoxin level was less than 10

endotoxin units (EU) per g of PHB irrespective of the bacterial strains employed and the PHB

content in the cell. Therefore the endotoxins in the PHB-A used in our study is most probably

negligible. Nevertheless, it would be interesting to separate the effect of the bacterial cell

wall from PHB as such. It might not be easy to obtain PHB-A in a pure form. A density

gradient centrifugation protocol has been described to isolate PHB, obtaining amorphous

PHB with high purity (Nickerson, 1982). So it would be worthwhile to follow this possibility. It

amorphous PHB could be obtained in this way with no contamination of other cellular

polymers, such amorphous PHB could be of great help in unraveling the mode of action of

PHB. Alternatively, we can evaluate the (interfering) effect of the cell wall by using Ralstonia

eutropha with different PHB concentrations. This is only an approximation since the cell wall

composition is most probably also affected by the different culture conditions that lead to

different PHB cell concentrations.

6. The mode of action of poly-β-hydroxybutyrate in bivalve mollusk should be further

investigated. This research should focus on its regulating effect on the microbial community

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Chapter 6 General discussion

146

composition in the gastrointestinal tract of the animal, using different PHB-A concentrations.

Microbial community analysis could be done on 16S rDNA by DGGE or by high throughput

sequencing technology.. The latter technology has the advantage of generating a much more

detailed picture of the microbial community composition, depending on the “sequencing

depth”. However, the downside of this method is the price of the analysis (although falling)

and the overload of data that needs to processed and interpreted by specialists.

7. A combination of various immune-stimulating compounds (such as beta glucan) could

further enhance the pre-biotic effect of PHB on bivalve larvae.

8. The application of PHB can be considered in bivalve hatcheries industry. It is anticipated

that the necessary microbial biomass does not need to be added alive and rather small

amounts need to be dosed. This makes the application of PHB in hatcheries economically

feasible provided positive effects can be obtained.Research would still need to be done on

e.g. dose, frequency of application and strains.

9. In an attempt to link virulence factor production with the observed in vivo mortality,

challenged tests could be designed that compare the effect of the “wild type” pathogen with

mutant pathogen that lack certain putative virulence factors and eventually such mutant

that contain this virulence gene on a single copy plasmid (provided such strains can be

engineered for the pathogenic strains that have been used in this study. This experimental

approach could yet a much better insight into the importance of certain virulence factors in

the pathogenesis.

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REFERENCES/A

SUMMARY/SAMENVATTING/B

EXTRA-EXPERIMENT/C

ACKNOWLEDGMENTS/D

CURRICULUM VITAE/E

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APPENDIX –A References

176

Zaidi, C., Tukimat, L., Muzzneena, A. & Mazlan, A. (2003) Effects of egg density during

incubation on the quality and viability of mussel larvae, Mytilus edulis L. Pakistan

Journal of Biological Science, 6, 1041-1045.

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APPENDIX B Summary / Samenvatting

177

SUMMARY

The blue mussel (Mytilus edulis) is an important species in Europe, not only as a model for

environmental studies but also as aquaculture species. For this reason, the blue mussel was

considered an ideal bivalve model species for this study. One of the main problems in the

culture of bivalve mollusks are the repetitive episodes of mortality which seriously reduce

the commercial production as illustrated by the phenomenon of summer mortality (SM) in

juvenile oysters Crassostrea gigas in France, and brown ring disease (BRD) in adult carpet

shells (Ruditapes semidecussatus) in Spain. Not only the grow out production but also

bivalve hatcheries are hit by production losses. Disease outbreaks during larval stages are

often caused by Vibrionaceae as primary agents. Traditional methods to combat bacterial

diseases such as enhanced water treatment with UV and ozone or the use of antibiotics

proved to be insufficient, costly and in the case of antibiotics even compromising for the

environment and human health. In the last decade, a lot of research has been dedicated to

finding alternative methods to control disease outbreaks. Several studies have

demonstrated that the use of poly-β-hydroxybutyrate (PHB) might constitute an ecological

and economical, sustainable alternative to fight infections in aquaculture. PHB is the most

common polyhydroxyalkanoate that is accumulated as carbon and energy reserve by a wide

variety of prokaryotes and may account for 90% of the cellular dry weight in some specific

bacteria, e.g., Ralstonia. This PhD research is the first study to evaluate the use of PHB as an

innovative production strategy to improve the performance of bivalve larvae. The following

three approaches were used:

(1) First, the application of PHB in mussel larvae cultures in crystalline form (PHB-C: i.e.

extracted from the bacterial cells) and amorphous form (PHB-A: i.e. still contained in the

bacterial cells) was compared. PHB was supplied at concentrations of 0.1 mg L-1, 1.0 mg L-1,

and 10.0 mg L-1 to the culture water of the larvae, starting 2 days after fertilization. The

results indicated that PHB-C was ineffective while the supplementation of 1 mg L-1 PHB-A

combined with microalgae significantly increased the survival. It did not, however, stimulate

the growth of the larvae and had no beneficial effect on the metamorphosis process. The gut

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APPENDIX B Summary / Samenvatting

178

microbiota of the mussel larvae was not significantly modulated by PHB supplementation in

terms of diversity and abundance and, therefore, could not be related to the larval

performance. Upon further investigation, a total of 22 PHB-degrading isolates were

identified from the mussel larvae fed with either crystalline PHB or amorphous PHB. 16 of

which belonged to the genus Pseudoalteromonas. Hypothesizing better survival and growth

by adding a strong PHB-degrading bacterium to the mussel culture in combination with

amorphous PHB, the isolate ARC4B5 was administered at a concentration of 106 CFU mL-1,

but the strain surprisingly suppressed larval growth.

(2) Secondly, the possible antimicrobial effect of PHB and its monomer in mussel larvae was

investigated. Their protective effect against two pathogenic bacteria Vibrio splendidus and

Vibrio coralliilyticus was studied both in vitro and in vivo at pH7 and pH8. pH-dependency

can be explained by the fact that fatty acids can pass the cell membrane only in their

undissociated form, which is more prominent at lower pH. For the in vitro tests, the

monomer β-hydroxybutyrate (β-HB) was used since bacteria can degrade PHB into β-HB

which is known to have antimicrobial activities. Only the highest concentration (125 mM)

significantly decreased the growth of the pathogens compared to the other concentrations

at both pH levels, except for V. coralliilyticus at pH8. The influence of β-HB on the virulence

factors of the pathogens was dependent on the dosage, Vibrio species, and pH. A clear

pattern could not be distinguished. The highest β-HB concentration (125 µM) inhibited some

virulence factors of the pathogenic bacteria such as hemolysis, phospholipase, but also

stimulated caseinase in V. splendidus at pH7 and V. coralliilyticus at pH8. In addition, β-HB

had no effect on gelatinase, lipase, and biofilm formation. The concentration of 125 µM is

biologically speaking irrelevant for mussel larvae and hence the results should be put in the

right perspective

Concerning the in vivo tests, survival of mussel larvae that received PHB-A at a

concentration of 1 mg L-1 PHB-A 6h before the challenge, was higher compared to the

survival of the larvae that received the PHB-A pre-treatment 24h before the challenge.

Larvae that received PHB-A 6h before the challenge showed a 55% and 25% increase in

survival compared to the controls when measured 96h after exposure to the pathogens V.

splendidus and V. coralliilyticus respectively.

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APPENDIX B Summary / Samenvatting

179

(3) Finally, the impact of amorphous poly-β-hydroxybutyrate (PHB-A) (1mg PHB-A L-1) on

gene expression of the antimicrobial peptides (AMPs) mytimycin, mytilinB, defensin and the

hydrolytic enzyme lysozyme in exposed mussel larvae was looked into during in vivo

challenge tests with V. coralliilyticus (105 CFU mL-1). Phenoloxidase (PO) activity in the

challenged mussel larvae was also evaluated. RNAs were isolated from mussel larvae tissue,

and AMPs were quantified by q-PCR using 18SrRNA gene as a housekeeping gene. The

results of this study demonstrated PHB-A regulated immune gene expression in mussel

larvae, as revealed by PO activity in unchallenged and challenged (48h after exposure)

larvae. This may at least partly explain why larval survival increases in the presence of PHB-

A.

It was demonstrated that 2 day-old blue mussel larvae activate the expression of the three

AMPs, mytimycin, mytilinB and defensin upon invasion by the pathogen V. coralliilyticus. The

level of expression is up- and down-regulated in the first 12hours after exposure in an

unclear pattern, but rapidly increases from 24 to 48 h post-exposure. The expression of

lysozyme, however, remains very low and stable during the first 48 h after challenge. The

mytimycin gene is one of the genes with the strongest expression in blue mussel larvae.

In conclusion, it can be stated that the biological compound PHB supplied as freeze-dried

Ralstonia can bring added value to bivalve larvae culture by its ability to fight pathogenic

infections and stimulate immune gene expression of these species. The fact that PHB-A

seems to have a positive role opens also the possibility to apply microbial biomass rich in

PHB rather than PHB-C, which needs to be chemically extracted, increasing the price of its

production. A thorough validation of the benefits of PHB-A at a commercial hatchery level

might provide the necessary data to verify the economic feasibility of PHB-A application as a

disease controlling tool.

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APPENDIX B Summary/Samenvatting

181

SAMENVATTING

De blauwe mossel (Mytilus edulis) is een belangrijke soort in Europa, niet alleen vanuit

ecologisch standpunt maar ook als aquacultuur soort. Dit is de reden waarom de blauwe

mossel ook voor deze studie als model heeft gediend voor de klasse van de bivalven. Een

van de belangrijkste problemen waarmee de sector te kampen heeft, zijn de recurrente

periodes van mortalteit die de commerciële productie zware slagen kan toedienen zoals

geïllustreerd door het fenomeen van zomermortaliteit bij juveniele holle oester Crassostrea

gigas in Frankrijk and de bruine ringziekte bij adulte tapijtschelpen (Ruditapes

semidecussatus) in Spanje. Niet alleen de schelpdierproducenten maar ook de broedhuizen

van schelpdieren worden geconfronteerd met zware verliezen. Ziektes gedurende het larvale

stadium worden vaak veroorzaakt door Vibrionaceae als primaire vectoren. Traditionele

methoden om deze bacteriële ziekten te bestrijden zoals het doorgedreven zuiveren van het

water met UV en ozon of het gebruik van antibiotica blijken niet zo efficiënt te zijn, kostelijk

en bovendien, in het geval van antibiotica, zelfs compromitterend voor het milieu en de

menselijke gezondheid. In het laatste decennium, is veel onderzoek gewijd aan het

ontwikkelen van alternatieve methodes om het uitbreken van ziektes in de hand te houden.

Verschillende studies hebben aangetoond dat het gebruik van poly-β-hydroxybutyrate (PHB)

een ecologisch en economisch verantwoord alternatief kan betekenen om infecties in

aquaculture te bestrijden. PHB is de meest voorkomende polyhydroxyalkanoate dat als

koolstof en energie reserve wordt opgeslagen door een wijd gamma aan prokaryoten, tot

zelfs 90% van het droog celgewicht in specieke bacteriële stammen zoals Ralstonia. Dit

doctoraatsonderzoek heeft voor de eerste keer de applicatie van PHB in mollusken en dus

ook in blauwe mossellarven geëvalueerd. Drie strategieën werden gevolgd:

(1) Als eerste werd de toediening van PHB in het kweekwater van mossel larven onder

crystallijne vorm (d.i. geëxtraheerd uit de bacteriële cellen, PHB-C)) en amorfe vorm (d.i. nog

ingecapseld in de gevriesdroogde bacteria, PHB-A) met elkaar vergeleken. PHB werd

toegediend in concentraties van 0,1 mg L-1, 1,0 mg L-1 and 10,0 mg L-1, startend 2 dagen na

de bevruchting van de eitjes. De resultaten toonden aan dat PHB-C geen impact had terwijl

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APPENDIX B Summary/Samenvatting

182

de supplementatie van 1,0 mg L-1 PHB-A in combinatie met microalgen leidde tot significante

verhoging van de larvale overleving. Er was echter geen effect op de larvale groei noch op

het proces van de metamorphose. De samenstelling en rijkdom van de intestinale microbiële

flora van de mossellarven was niet significant gewijzigd door de toediening van PHB en kon

dus niet gerelateerd worden aan de larvale performantie. In een volgende stap werden in

het totaal 22 PHB-afbrekende isolaten gekarakterizeerd afkomstig van mossel larven die

PHB-C of PHB-A waren toegediend. Alle isolaten behoorden toe tot het genus

Pseudoalteromonas. De hypothese dat de toevoeging van een sterk PHB-afbrekende

bacterium een positieve invloed zou hebben op overleving en groei van de mossellarven

wanneer die toegevoegd werd aan het kweekwater samen met PHB-A, werd ontkracht

gezien de toevoeging van ARC4B5 aan 106 CFU mL-1 de larvale groei zelfs onderdrukte.

(2) Vervolgens werd de mogelijke anti-bacteriële werking van PHB en het afgeleid

monomeer product onderzocht. Hun beschermende werking tegen twee pathogenen voor

bivalven, Vibrio splendidus en Vibrio coralliilyticus werd zowel in vitro als in vivo uitgetest bij

pH7 en pH8. De pH afhankelijkheid kan verklaard worden door het feit dat de vetzuren

alleen in ongedissocieerde vorm doorheen de celwand kunnen passeren en deze vorm is

meer vertegenwoordigd bij lage pH. Voor de in vitro tests werd de monomeer β-

hydroxybutyrate (β-HB) gebruikt omdat bacteriën PHB degraderen naar β-HB dat bewezen

anti-microbiële activiteiten heeft. Alleen de hoogste concentratie (125 mM) inhibeerde

significant de groei van beide pathogenen onafhankelijk van de pH, met uitzondering van V.

coralliilyticus by pH8. De impact van β-HB op de virulentie factoren van de pathogenen was

afhankelijk van de concentratie, Vibrio soort en pH. Er kon geen duidelijk patroon

waargenomen worden. De hoogste β-HB concentratie (125 µm) inhibeerde sommige

virulentie factoren van de pathogenen zoals haemolyisis and phospholipase maar

stimuleerde ook de productie van caseinase in V. splendidus bij pH7 en V. coralliilyticus bij

pH8. Het gebruik van β-HB had geen effect op gelatinase, lipase en biofilm vorming. De

concentratie van 125 µM is biologisch gesproken echter irrelevant voor mossellarven en de

resultaten moeten dus voorzichtig geïnterpreteerd worden.

In de in vivo testen werd duidelijk dat de overleving van de mossel larven die PHB-A (1,0 mg

L-1 ) 6 hr voor de challenge toegediend kregen, hoger was dan de overleving bij larven die

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APPENDIX B Summary/Samenvatting

183

PHB-A 24hr voor de challenge kregen. In vergelijking met de controle dieren, vertoonden

deze eerstgenoemde larven een toename in overleving van 55% en 25% na 96h blootstelling

aan de pathogenen V. splendidus en V. coralliilyticus respectivelijk.

(3) Tenslotte werd de impact van PHB-A (1,0 mg L-1 ) op de genexpressie van de

antimicrobiële peptides (AMPs) mytimycin, mytilinB, defensin en van het hydrolytisch

enzyme lysozyme bestudeerd tijdens in vivo challenge tests met V. coralliilyticus (105 CFU

mL-1). Phenoloxidase activiteit (PO) in de larven werd ook gemeten. RNA werd geïsoleerd

van gehomogeniseerde mossel larven en de expressie van de AMPs werd gekwantificeerd

met q-PCR en met 18SrRNA gen als huishoudgen. Uit deze studie komt naar voor dat PHB-A

de immuun genexpressie in larven kan regelen gezien de PO activiteit zowel in de controle

als blootgestelde larven toeneemt na 48h. Dit zou deels kunnen verklaren waarom de larvale

overleving toeneemt wanneer PHB-A wordt toegediend aan het kweekwater.

Er is aangetoond dat larven van 2 dagen-oud de expressie van drie AMPs, mytimycin,

mytilinB en defensin activeren bij een invasie van de pathogeen V. coralliilyticus. Het

expressie niveau gaat op en neer in de eerste 12 uren na blootstelling zonder patroon, maar

stijgt snel na 24 tot 48 uur blootstelling. De expressie van lysozyme echter blijft heel laag en

stabiel gedurende de eerste 48 uur na blootstelling. Het mytimycin-gen is een van de genen

die het sterkst tot expressie komt in blauwe mossellarven.

Als besluit van deze studie kan gesteld worden dat PHB, aangeleverd in de vorm van

gevriesdroogde Ralstonia een toegevoegde waarde kan bieden voor larvale cultuur van

bivalven dankzij zijn directe werking op de pathogeen en stimulerend effect op de expressie

van immuun-gerelateerde genen. Dit opent de mogelijkheid om microbiële biomassa rijk aan

PHB te gebruiken eerder dan PHB-C dat eerst chemisch geëxtraheerd moet worden wat de

productiekost opdrijft. Een grondige validatie van de voordelen van PHB-A op het niveau van

een commercieel broedhuis kan de nodige data aanleveren om de economische

haalbaarheid van het gebruik van PHB-A als ziekte controlerend middel te evalueren.

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APPENDIX C Extra – experiment

185

EXTRA- EXPERIMENT

Below are the results of an experiment supplementary to the results already shown in fig 4.2

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APPENDIX C Extra – experiment

186

Figure 4.2. (Repeat experiment). Survival of blue mussel challenge with V. coralliilyticus and

V. splendidus at 105CFU mL-1 under different experimental condition.

........................................................................................................................................................................................................................................................................ !

~ i

EL. ~ -----x "i 60 '\ I ------s-... ____

8 ~ 40 \ !

0 24 48 72 96

Post exposurejh)

·····+ ··· Control ---0-- V. splendidus

-I'HB - ->f - V. splendidus + I'HB

Challenge test z. 1 mg PHB L-1 ; Z4h

100

_80

ê60

~40 l 20

-~ ' '

...

\ \

\

0 J.._ ______ -"'0:::-:=-",-=-~----~

0 24 48 72 96

Post exposure jh)

·····+ ····· Control ---8-- V. splendidus

____.._.. I'HB - ->f - V.splendidus+ I'HB

Challenge test 3. 10 mg PHB L·1; 6h

100

20 0 J.._ ______________ _

0 24 48 72 96

Post exposure lh)

·····+ ····· control ---8-- V. splendidus

---+-PHB - -::+ - V. splendidus + PHB

Challenge 4. 10 mg PHB L·1; Z4h

100

80

20

0

0 24 48 72 96

ut ' i 20 \ i o - a -s l

0 24

·····+ ····· Control

____.._.. I'HB

48 72 96

Post exposurelh)

---e--- V. coralliilyticus

- -;~- V. coralliilyticus+ I'HB

: i

I i

I i i

·----··· ... • • i '\ i

100

40 '\ ! 2: _L_ _______ \_\===-!~[+- !

0

·····+ ···· Control

____.._.. I'HB

24 48 72 96 i ;

Post exposure lh) ! ;

---8-- V. coralliilyticus 1 i

- ->f - V. coralliilyticuS+ I'HB ~

i i

~ ,: .,. I l 60 ' ;

l : _L_ _______ '_\_\--"'~L-:::-:::-~-=- ti'---- I 0 24 48 72 96 !

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·····+ ···· Control ---8-- V. coralliilyticus ~ - -::+ - V. coralliilyticus .. I'HB i

i i

'\, i m '\\.._______ I

0 L_ __________ ~=~=~=~-~ i

100

0 24 48 72 96 ! ;

Post exposure (h) I hl i Post exposure i ···+ ··· Control ---8-- V. splendidus ·····+ ···· Control ---e-- V. coralliilyticus !

____.._.. I'HB - ->f - V. splendidus .. I'HB ____.._.. I'HB ---><:--- V. coralliilyticu<-> I'HB ~ .................................................................................................................................................................................................................................................................... .!

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APPENDIX –D Acknowledgement

187

ACKNOWLEDGEMENTS

First of all, I am very grateful to the Vietnamese government (The Vietnam International

Education and Development, and Aquaculture Biotechnology program of Ministry of

Agriculture and Rural Development) and the VLIR-OI project ZEIN2011PR389 (University of

Ghent, Belgium) for their financial support in completing my PhD research program.

For over more than four years, this dissertation would not have been possible without the

guidance and help of several individuals who have contributed directly or indirectly to bring

this thesis to a successful end.

My special thanks goes to my promotor Prof. dr. ir. Peter Bossier, for his kind support and

overall guidance in the completion of my PhD. He was always available during his very busy

schedule to give me useful feedback, suggestions and corrections in every aspect related to

my PhD work. I considered myself very lucky to have had the chance to work under the

supervision of such a kind person and a well-kown professor in his field.

My deepest gratitude goes to Dr. ir. Nancy Nevejan, for her guidance and especially for her

patience in correcting my papers and the manuscript of this thesis. I would also like to thank

her for her support not only in my scientific work but also for my family situation. I will never

forget you, together with your husband Marc, looking for a solution to help my small son,

where you introduced us and explained all to the doctor in an Antwerp hospital for the

Cochlear implant for my small son.

A special thank you goes to Dr. Peter De Schryver, who has always stood by me, from

designing the scientific experiments to the practical work in the beginning of my PhD. He has

been contributing to all the papers during my study. I also want to thank him for his patience

during the corrections of my papers.

I am very grateful to Dr. Ana Bossier for her support and critical thesis correction. I also wish

to thank her for the suggestions and comments on the manuscript. Many thanks for the

home made cakes and your willingness to look after of me when I got sick. Thank you very

much!

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APPENDIX –D Acknowledgement

188

I also wish to express my deep gratitude to Em. prof. dr. Patrick Sorgeloos, for giving me an

opportunity to study and do research in both the international master program and the PhD

degree at the Laboratory of Aquaculture & Artemia Reference Center, University of Ghent,

Belgium. Thank you very much.

I would like to thank all the members of the examination and reading committee. Thanks a

lot for sharing your expertise and knowledge. I am very grateful for your critical comments

and suggestions towards improving the quality of this manuscript.

I am also grateful to all the staff at the Research Institute for Aquaculture #3: former director

Prof. Dr. Nguyen Chinh, Assoc. Prof. Dr. Nguyen Thi Xuan Thu, Dr. Dao Van Tri, Dr. Nguyen

Viet Nam, Dr. Truong Ha Phuong, Dr. Vo The Dung, Mr. Nguyen Co Thach, Mr. Pham Vu Hai

and current director Dr. Nguyen Huu Ninh for always supporting me, giving me the

opportunity to study abroad, and encouraging me during my study period. Also, my

particular thanks goes to my close family friends at home, Binh Hieu, Phong Vi, Thoa Chinh,

Hien Thien, Tam Phuc, Phuong Tinh. You have always been helpful and encouraged my

family when I was not at home. Thank you very much!

My moments at ARC with my colleagues will be cherished forever. I would like to give all of

you a great thank you: Marc Verschraeghen, Tom Baelemans, Alex Pieters, Caroline Van

Geeteruyen, Mathieu Wille, Jorg Desmyter, Brigitte Van Moffaert, Anita De Haese, Geert Van

de Wiele, Christ Mahieu, Prof. dr. Gilbert Van Stappen, Jean Dhont, Kristof Dierckens,

Margriet Drouillon, Kenny Vandenbroucke, Michael Yufeng Niu, Kartik Baruah and Parisa

Norouzitallab. Thank you for always being available to help me during my lab work at ARC.

Especially, to the mussel team Mieke Eggermont and Aäron Plovie, thank you for your kind

help and always accompanying me during the experiments with our lovely mussel larvae. To

all ARC staff members, thanks for the good friendship and assistance. I think I will miss all of

you.

Many thanks to Dr. Tinh, who recommended me the ARC before I started my PhD

application and to Dr. Phuoc for his continuous support, suggestions and scientific

comments on the manuscript. Special thanks to my PhD colleagues: XHong, VDung, Toi, Van,

Tho, Joseph, Gladys, Eamy, Bai Nan, Lenny, Sofie, Hu Bing, Li Xuan, Ramin, Mohamed,

Spyros, Bay, Loan, Manh, Dipesh, Sony, Rajeish, Duy, Ruy, Navid, Bárbara and Thai, thanks

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189

for sharing the secret experience, co-operation, and science discussions. I wish you all the

best!

To the Vietnamese people and students who are living Belgium: Tam Anh, Thu Ha, Xuan

Hong, Viet Hong, Dung, Giang, Thuc, Nguyen, Phuc, Loc, Toi, Tuyen, Hanh Tien, Tuan, Luong,

Linh, Duy, Ha, Phuong, Cuong, Ngoc, Thai, and family Giang Duc, Phuong Thuong, Thuy Khai,

Han Dung, Ha Tu, Thuy Anh, Quan Ut, Tho Ut, Trang Quan, thanks for a nice friendship and

social activities that made my life much more colorful during my stay in Ghent.

To my master students Tam and Christine Ludevese for their support and assistance during

extensive periods of the experiment. Thank you!

I was lucky to live in OBSG, a beautiful and friendly place for students from developing

countries who study in Gent. I would like to thank Father Charles de Hemptinne, and all the

staff for all their service and the accommodation during my stay in Ghent.

Great thanks to all members of my family: my mother, brothers and sisters, my mother in

law, brothers in law, and my sisters in law who have always supported me and expected me

to finish my PhD degree. I know that words are not enough to express my gratitude to them.

This thesis is a present for them. Thank you.

I am especially thankful to my foreign family Heidrun Inger Wergeland and Ian Mayer for

their continuous encouragement and contribution during my study. I do not know how to

express my gratitude for what you have done for me. Thank you.

Finally, I would like to dedicate this thesis to my lovely wife Thanh Thuy, my daughter Anh

Thi and my small son Anh Khoi for their support, encouragement and their patience during

the 4 years of my PhD research. My wife has worked hard to take care of our children and

has suffered a lonely life during the time I have not been at home. Behind the success of my

PhD research as well as the success of my life were always her silent sacrifices. And I would

like to say sorry to my little son for leaving him when he was only 2 weeks old until now.

Thank you very much!

Ghent, 02/05/2016

Hung

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191

CURRICULUM VITAE

A. PERSONAL DATA

Full name: Nguyen Van Hung

Day of birth: March 12, 1973

Place of birth: Binh Dinh, Viet Nam

Nationality: Vietnamese

Organization: Mariculture Research and Development Center, Research Institute for

Aquaculture No.3, Ministry of Agriculture and Rural Development, Viet Nam.

Position: Researcher/ Scientific staff

Head office: 33 Dangtat, Nhatrang, Vietnam

Telephone: + 84.58 3831 138, Fax: + 84.58 3831 846

E-mail: [email protected] / [email protected]

B. EDUCATION

2011 – 2016: PhD in Applied Biological Science at Laboratory of Aquaculture & Artemia

Reference Center, Faculty of Bioscience Engineering, University of Ghent, Belgium.

2003 – 2005: Master of Science in Aquaculture, University of Ghent, Belgium

1993 – 1998: Aquaculture Engineering, Nhatrang University of Fisheries, Vietnam.

C. PROFESSIONAL ACTIVITIES

2009–2014: Vice-director of Mariculture Research and Development Center.

2007-2010: National Project: Preservation of the gene pool of Aquaculture marine species in

Vietnam (Coordinator).

2008: Institute project: The use of probiotic in marine fish parramundi (Lates calcarifer) and

orange- spotted grouper (Epinephelus coioides) larvae and fingerling (Coordinator).

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2008-2010: National project KC.06/06-10: Research on seed production and grow out the

technology of three economic value marine fish (Co-coordinator).

2008-2010: National project KC.06/06-10: Research on seed production and grow out the

technology of three coral ornamental fish to export (Co-coordinator).

2007: Cooperation project between Vietnam-Russian tropical Center- Moscow University

and Research Institute for Aquaculture no.3. Project Title: Induced Oocyte maturation and

gamete quality in coral reef fish species with alternative reproductive strategies

(Coordinator).

2006-2008: Cooperation project (JICA and RIA3): Economic marine finfish Research and

Development: Broodstock management, Implantation for hormone stimulation to spawning,

Biological reproduction, larval rearing, and live food (Group leader/ Scientific staff).

2002-2003: Cooperation project with ICLARM (World fish center) Project title: Seed

production, nursery rearing and grow out of Sea-cucumber Holothuria scabra (Staff

member).

2000-2001: National project: Reproduction and grow out of Abalone Haliotic asinina (Staff

member).

1998-1999: National Project: Seed production technology and grow out of Yellow pearl

oyster Pinctada maxima Jameson, 1901 (Staff member).

D. PUBLICATIONS & INTERNATIONAL CONFERENCES

Publication in international peer-reviewed journals

Hung, N. V., De Schryver, P., Tam, T. T., Garcia-Gonzalez, L., Bossier, P. & Nevejan, N. 2015.

Application of poly-β-hydroxybutyrate (PHB) in mussel larviculture. Aquaculture 446(0) p:

318-324.

International conferences

Hung, N.V., N. Nevejan., P. De Schryver., Dung, N.V., P. Bossier 2016. Poly-ß-hydroxybutyrate

(PHB) regulates the immune response in mussel larvae challenged with Vibrio coralliilyticus.

European Marine Science Educators Association 2016 Conference 2016: VIVES, Brugge,

Belgium (Poster presentation)

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Hung, N.V., P. De Schryver, T. T. Tam, L. Garcia-Gonzalez, P. Bossier and N. Nevejan, 2013.

Effect of Poly - β - hydroxybutyrate on bivalve larvae culture. World Aquaculture Society

Conference, Ho Chi Minh City, Vietnam (Oral presentation).

Hung, N.V., P. De Schryver, T. T. Tam, P. Bossier and N. Nevejan, 2013. The use of poly - β -

hydroxybutyrate in bivalve larviculure. Larvi 2013 Conference, 6th fish & shellfish larviculture

symposium, Ghent Belgium (Poster presentation).

Hung, N.V., D.A. Pavlov, Nata’ya G. Emel’yanova and Thuan, L.T.B. 2007. Induced oocyte

maturation and gamete quality in coral reef fish species with alternative reproductive

strategies. Asia Pacific Aquaculture conference in Ha Noi, Viet Nam (Poster presentation).

Hung, N.V., Tri, D. V, Norihisa, I. 2007. Spawning of Coral trout grouper Plectropomus

leopardus in Nha Trang, Viet Nam. Asia Pacific Aquaculture conference in Ha Noi, Viet Nam

(Oral presentation).

Publications at national level

Thoa, N.T., Thuy, N.T.T., Hung, N.V., 2015. The first studies of several common diseases and

the prevention of diseases in the reproduction of frog crab (Ranina ranina Linnaeus, 1758).

Journal of Agriculture and Rural Development (6), p: 101-107 (Abstract in English).

Thuy, N.T.T., Thoa, N.T., Phuong, D.T., Hung, N.V., 2014. Reproductive biology of the red frog

crab (Ranina ranina Linnaeus, 1758) from Khanh Hoa province, Vietnam. Journal of

Agriculture and Rural Development (5), p: 77-85 (Abstract in English).

Hung N.V., Binh D.T., 2010. Application of mtDNA to evaluate the population diversity of

coral trout grouper (Plectropomus leopardus) distributed in Vietnam coastal area. Journal of

Biotechnology, Nha Trang University (Abstract in English).

Hung N.V., Bon. L. Q, 2008. Research population genetic on coral trout grouper in Khanh Hoa

and Hai Phong coastal area. Fisheries Technological science No.6/2009 ISN 1859 -1299 p: 12-

14 (Abstract in English).

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Hung N.V., Bon. L. Q, Dung.N.V and Ishibashi N., 2009. Research on some reproduction

biology of coral trout grouper (Plectropomus leopardus) in Khanh Hoa coastal area.

Agriculture publishing house HCM, Vietnam p: 445 – 453 (Abstract in English).

Hung N.V., N.T.T.Hien, V.T.T. Em, 2009. Research on food and nutrition demand of coral

trout grouper (Plectropomus leopardus) from larvae rearing to fingerling stage. Agriculture

publishing house HCM, Vietnam (Abstract in English).

Hung N.V., D.T. Phuong, V.T.T. Em, N.D.Tinh, 2009. The effects of some probiotic on growth

and survival rate in larvae rearing or orange spot grouper (Epinephelus coioides) and seabass

(Lates calcarifer). Agriculture publishing house HCM, Vietnam (Abstract in English).

Hung N.V., L.Q. Bon, H.T. Ngoc, 2009. Status of gene pool preservation of aquaculture

marine, brackish species. Agriculture publishing house HCM, Vietnam (Abstract in English).

Hung N.V., Tra. N.T.P; Anh. N.T.K, 2008. Research on some biological characteristic of

damselfish (Dascyluss trimaculatus). Agriculture publishing house HCM, Vietnam p: 454-464

(Abstract in English).

Hung, N.V., Phuong, D.T., Thuy, T.T., 2009. Evaluate fertility of male, white shrimp

(Litopenaeus vannamei) F1 generation derived from Hawaii. Agriculture publishing house

HCM, Vietnam, p: 822 – 832 (Abstract in English).

Thuy, N.T.T., Hung, N.V., Thuy, T.N., 2009. Detecting parasites and bacteria in damselfish

(Dascyllus trimaculatus) cultured at Nhatrang with the exophthalmia and ulcerative

infection. Agriculture publishing house HCM, Vietnam, p: 694 – 702 (Abstract in English).

Hung, N.V., Bon, L.Q., Duy, V.D., Ngoc, H.T., Minh, D.M., 2008. Some classification and

population genetic features of Brown- marbled grouper (Epinephelus fuscoguttatus,

Forssakal 1775) in Khanh Hoa coastal area. Fisheries Technological Science No. 9/2008 ISSN

1859 -1299 p: 32 – 34 (Abstract in English).

E. DOCTORAL TRAINING SCHOOL / PROFESSIONAL TRAINING COURSES

Specialist courses

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- Applying Chemical Tools to study Biology, held by VIB, University of Antwerp,

Belgium.

- Basis of biology for engineers, held by IMEC, University of Leuven, Belgium.

- Microbial Community Management in Aquaculture, held by ARC, Ugent, Belgium.

Transferable skills

-Personal Effectiveness held by Doctoral Traning School, Ugent, Belgium.

-Project management held by Doctoral Traning School, Ugent, Belgium.

-Scientific writing skill, held by Doctoral Training School -VIB, Ugent, Belgium.

-Effective scientific communication held by Doctoral Traning School, Ugent, Belgium.

-Advanced Conference Skills in English held by Doctoral Traning School, Ugent,

Belgium.

Professional training courses

2009: Practice on molecular biotechnology held by Biotechnology Institute, Ha Noi, Vietnam.

2007: Grouper Hatchery Training course funded by Network of Aquaculture Centers in Asia –

Pacific (NACA). Held at Brackishwater Aquaculture Development Center- Situbondo,

Research Institute for Mariculture, Gondol, Indonesia.

2006: Intensive Aquaculture production, Environmental, considerations & Food safety. Held

at Ministry of Agriculture and Rural Development, Center for International

Agriculture Development Cooperation Cinadco, Israel.

2003: Babylon Seed production technology funded and held by Ocean Wave Seafood,

Melbourne, Australia.

1999: Taxonomy and diversity of Tropical marine mollusk held by DANIDA, Denmark.

G. THESIS SUPERVISION

1. Tran Thanh Tam, 2013. The use of poly-β-hydroxybutyrate to increase robustness in blue

mussel larviculture. Master of Sciences in Aquaculture at the Faculty of Bioscience

engineering, Ghent University, Belgium.

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196

2. Christine Llorente Ludevese, 2015. Antimicrobial effect of poly-β-hydroxybutyrate (PHB)

on blue mussel larviculture. Master of Sciences in Aquaculture at the Faculty of Bioscience

engineering, Ghent University, Belgium.